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Sample records for fluorescence reverse transcription-polymerase

  1. Interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction as a diagnostic aid for synovial sarcoma.

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    Shipley, J; Crew, J; Birdsall, S; Gill, S; Clark, J; Fisher, C; Kelsey, A; Nojima, T; Sonobe, H; Cooper, C; Gusterson, B

    1996-02-01

    Identification of the t(X;18)(p11.2;q11.2) that is associated with a high proportion of synovial sarcoma can be a useful diagnostic aid. The translocation results in fusion of the SYT gene on chromosome 18 to either the SSX1 or the SSX2 gene, two homologous genes within Xp11.2. Two-color interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction were assessed as approaches to identify the rearrangement in well characterized cases. The presence of the translocation, and the specific chromosome X gene disrupted, were inferred from the configuration of signals from chromosome-specific centromere probes, paints, and markers flanking each gene in preparations of interphase nuclei. Rearrangement was found in two cell lines and eight of nine tumor samples, including analysis of five touch imprints. This was consistent with cytogenetic data in four cases and reverse transcription polymerase chain reaction analysis using primers known to amplify both SYT-SSX1 and SYT-SSX2 transcripts. The transcripts were distinguished by restriction with LspI and SmaI. Contrary to previous suggestions, there was no obvious correlation between histological subtype and involvement of the SSX1 or SSX2 gene. These approaches could also be applied to the identification of tumor-free margins and metastatic disease.

  2. Microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse-transcription polymerase chain reaction for the rapid detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens.

    Science.gov (United States)

    Jia, Ruan; Chengjun, Sun; Heng, Chen; Chen, Zhou; Yuanqian, Li; Yongxin, Li

    2015-07-01

    Enterovirus 71 and Coxsackievirus A16 are the main pathogens causing hand-foot-mouth disease. In this paper, microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse transcript-polymerase chain reaction has been developed for the detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens. The specific reverse transcription-polymerase chain reaction amplicons labeled with SYBR Orange were separated by microchip capillary electrophoresis and detected by laser induced fluorescence detector within 7 min. The intraday and interday relative standard deviation of migration time for DNA Marker was in the range of 1.36-2.94 and 2.78-3.96%, respectively. The detection limits were as low as 2.06 × 10(3) copies/mL for Enterovirus 71 and 5 × 10(3) copies/mL for Coxsackievirus A16. No cross-reactivity was observed with rotavirus, astrovirus, norovirus, and adenovirus, which showed good specificity of the method. This assay was validated using 100 throat swab specimens that were detected by real-time reverse-transcript polymerase chain reaction in parallel and the two methods produced the same results. This study provided a rapid, sensitive and specific method for the detection of Enterovirus 71 and Coxsackievirus A16, which make a contribution to significant time and cost saving for the identification and treatment of patients.

  3. A reverse transcription-polymerase chain reaction assay for the detection of avian pneumovirus (Colorado strain).

    Science.gov (United States)

    Ali, A; Reynolds, D L

    1999-01-01

    A reverse transcription-polymerase chain reaction assay was developed for the detection of avian pneumovirus (Colorado strain) (APV-Col). The specific primers were designed from the published sequence of the matrix protein gene of APV-Col. The primers amplified a product of 631 nucleotides from APV-Col. The assay identified only APV-Col and did not react with Newcastle disease virus and infectious bronchitis virus.

  4. Typing of Poultry Influenza Virus (H5 and H7 by Reverse Transcription- Polymerase Chain Reaction

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    Cesare Bonacina

    2010-01-01

    Full Text Available The ability of the influenza Orthomixovirus to undergo to continually antigenically changes that can affect its pathogenicity and its diffusion, explains the growing seriousness of this disease and the recent epizoozies in various parts of the world. There have been 15 HA and 9 NA type A sub-types of the influenza virus identified all of which are present in birds. Until now the very virulent avian influenza viruses identified were all included to the H5 and H7 sub-types. We here show that is possible to identify the H5 and H7 sub-types with reverse transcription-polymerase chain reaction (RT-PCR by using a set of specific primers for each HA sub-type. The RT-PCR is a quick and sensitive method of identifying the HA sub-types of the influenza virus directly from homogenised organs.

  5. Diagnosis of Brazilian vesiculoviruses by reverse transcription-polymerase chain reaction

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    Daniela Wey Bonutti

    2005-04-01

    Full Text Available We describe a reverse transcription-polymerase chain reaction (RT-PCR and a nested-PCR for diagnosis of Piry, Carajás, Cocal, and Alagoas vesiculoviruses from Brazil. The RNA extracts of viral and clinical samples were submitted to a RT-PCR using Vesiculovirus G primers that amplify part of the glycoprotein gene. The RT-PCR produced amplicons of expected size, 290 base pair, for the four studied viruses. The RT-PCR showed a high sensitivity being 151.3 times (2.18 log more sensitive for the detection of Piry virus than the classical procedure for virus detection in tissue culture based on the viral cytophatic effect. Amplicons had nucleotides sequenced and were aligned in order to select internal primers for a nested-PCR to confirm the origin of Piry, Carajás, Cocal, and Alagoas Vesiculovirus. Ten blood and tarsal pad epithelial samples of infected Guinea-pigs had Vesiculovirus genome amplified by RT-nested-PCR.

  6. Detection of HCV-RNA by Reverse Transcription Polymerase Chain Reaction Using Biotinylated and Radioiodinated Primers

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    Ryu, Jin Sook; Moon, Dae Hyuk; Cheon, Jun Hong; Chung, Yoon Young; Park, Hung Dong; Chung, Young Hwa; Lee, Young Sang [Asan Medical Center, University of Ulsan, Seoul (Korea, Republic of)

    1994-07-15

    This study was performed to evaluate the clinical applicability of the reverse transcription polymerase chain reaction (RT-PCR) kit of HCV-RNA using biotinylated and radioiodinated primers. Study subjects were 118 patients with positive anti-HCV. HCV-RNA in patients serum was extracted by guanidium thiocyanate method. After first amplification, the product was reamplified by primers labelled with biotin and I-125. The final amplification product was detected by counting the radioactivity after incubation in avidin coated tubes. In 51 samples, the test was repeated for evaluation of reproducibility. This new method was also compared with conventional RT-PCR methods in 34 samples from patients with chronic liver disease. The results were as follows, 1) HCV-RNA was positive in 85(97%)of 88 patients with chronic liver disease, and in 23 (73%) of 30 patients with normal liver function. 2) In comparison with conventional method, HCV-RNA was detected in 32(94%) of 34 patients with new method, whereas in 27(79% ) of the same group with conventional method 3) Repeated test with new method in 52 samples demonstrated 82% of concordant result. In conclusion, new method with biotinylated and radioiodinated primers was more sensitive than conventional method. However, great care must be taken for quality control because there were considerable interassay variation and possibility of false positivity and false negativity.

  7. A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

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    Figueiredo Luiz Tadeu M

    1997-01-01

    Full Text Available We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

  8. 实时荧光RT-PCR方法检测香石竹环斑病毒%Detection of Carnation ringspot virus by real-time fluorescent reverse transcription polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    崔学慧; 陈舜胜; 于翠; 杨翠云

    2012-01-01

    本研究以香石竹环斑病毒(Carnation ringspot virus,CRSV)的5个分离物为研究对象,根据CRSV运动蛋白(MP)基因的保守序列设计一对特异性引物和TaqMan荧光探针,建立了检测CRSV的实时荧光RT-PCR(real-time fluorescem RTPCR)方法.该方法利用TaqMan探针水解产生的荧光信号实时监测目标基因的扩增,实现real-time fluorescent PCR扩增和检测同步进行.结果表明,本研究建立的实时荧光RT-PCR方法具有更快速、灵敏和特异的优点,与普通RT-PCR方法相比其灵敏度提高了100倍,适合于对进境种苗携带的CRSV的快速检测.%In this study, a pair of specific primers and a TaqMan probe were designed according to the conserved movement protein (MP) gene sequences of Carnation ringspot virus (CRSV), and a real-time fluorescent PCR method was applied for detection of CRSV. This method used the fluorescent signal generated from the hydrolysis of TaqMan probe to real-time monitor amplification of the target gene. It realized synchronization between real-time fluorescent RT-PCR amplification and detection. The results showed that the method was better than others in efficiency and specificity, and 100 times more sensitive than common RT-PCR. This method had potential to be applied in rapid detection of CRSV in incoming seedlings.

  9. Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3

    OpenAIRE

    Sharma, Deepa K.; Nalavade, Uma P.; Deshpande, Jagadish M.

    2015-01-01

    Background & objectives: The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. Methods: Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolat...

  10. Use of Existing Diagnostic Reverse-Transcription Polymerase Chain Reaction Assays for Detection of Ebola Virus RNA in Semen.

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    Pettitt, James; Higgs, Elizabeth S; Adams, Rick D; Jahrling, Peter B; Hensley, Lisa E

    2016-04-15

    Sexual transmission of Ebola virus in Liberia has now been documented and associated with new clusters in regions previously declared Ebola free. Assays that have Emergency Use Authorization (EUA) and are routinely used to detect Ebola virus RNA in whole blood and plasma specimens at the Liberian Institute for Biomedical Research were tested for their suitability in detecting the presence of Ebola virus RNA in semen. Qiagen AVL extraction protocols, as well as the Ebola Zaire Target 1 and major groove binder quantitative reverse-transcription polymerase chain reaction assays, were demonstrably suitable for this purpose and should facilitate epidemiologic investigations, including those involving long-term survivors of Ebola.

  11. Detection of Salmonella Typhi in blood with real time fluorescent quantitative reverse transcriptive polymerase chain reaction%利用实时荧光定量反转录-聚合酶链反应方法检测血液中伤寒沙门菌

    Institute of Scientific and Technical Information of China (English)

    樊粉霞; 娄静; 陈建才; 聂艳妮; 阚飙; 闫梅英

    2012-01-01

    Objective To establish an assay of real time fluorescent quantitative reverse transcriptive polymerase chain reaction (rRT-PCR) to detect Salmonella Typhi in blood. Methods Specific primers STY1631 gene of S. Typhi were designed and modified to establish rRT-PCR assay and the specificity and sensitivity of rRT-PCR with RNA as template were evaluated and verified by checking cultured Salmonella spp. in 51 serotypes, predominant non-salmonella enteric diarrheal pathogens and eight species of bacteria inducing bacteremia with fever as major symptom. The simulative blood specimens supplemented with S. Typhi were tested by the rRT-PCR assay. Results The established rRT-PCR assay successfully detected STY1631 gene of S. Typhi. Totally 48 S. Typhi isolates were amplified to be positive. Other isolates, including non-salmonella strains in 33 serotypes, predominant non-salmonella enteric diarrheal pathogens and eight species of bacteria causing bacteremia with fever, were amplified to be negative. For purified total RNA from pure cultured isolates, the detection limit of the assay was 1 pg per reaction, equal to 194 copies per reaction. The sensitivity achieved 1 × 102 cfu /ml with the purified nucleotide from simulative blood. Conclusion The rRT-PCR assay for detecting S. Typhi with high sensitivity and specificity was established, which would be suitable for the rapid diagnosis of S. Typhi infection and identification of other pathogens causing fever for the early warning, prevention and treatment of typhoid fever.%目的 建立实时荧光定量反转录-聚合酶链反应(real time fluorescent quantitative reverse transcriptionpolymerase chain reaction,rRT-PCR)方法检测伤寒沙门菌.方法 针对伤寒沙门菌STY1631基因设计特异性引物,通过优化反应条件,建立检测该靶基因的rRT-PCR方法,利用51个血清型的纯培养伤寒和非伤寒沙门菌菌株、常见非沙门致腹泻病原菌以及发热为主要症状的8种常见病原菌核糖

  12. The sensitivity and specificity of a reverse transcription-polymerase chain reaction assay for the avian pneumovirus (Colorado strain).

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    Pedersen, J C; Reynolds, D L; Ali, A

    2000-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of avian pneumovirus (APV), Colorado strain (US/CO), was evaluated for sensitivity and specificity. The single-tube RT-PCR assay utilized primers developed from the matrix (M) gene sequence of the US/CO APV. The RT-PCR amplified the US/CO APV but did not amplify other pneumoviruses, including the avian pneumoviruses subgroups A and B. The RT-PCR was capable of detecting between 10(0.25) mean tissue culture infective dose (TCID50) and 10(-0.44) TCID50 of the US/CO APV. These results have demonstrated that the single-tube RT-PCR assay is a specific and sensitive assay for the detection of US/CO APV.

  13. Detection of dengue virus serotype 3 by reverse transcription-polymerase chain reaction in Aedes aegypti (Diptera, Culicidae captured in Manaus, Amazonas

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    Valéria CS Pinheiro

    2005-12-01

    Full Text Available The detection of dengue virus serotypes from Aedes aegypti in Manaus, state of Amazonas was carried out using the reverse transcription-polymerase chain reaction technique. Fourteen pools out 82 (17.1% were positive for DENV3, providing a minimal infection rate of 2.1% of all analyzed infected female specimens of three different areas of the city.

  14. A Pipeline with Multiplex Reverse Transcription Polymerase Chain Reaction and Microarray for Screening of Chromosomal Translocations in Leukemia

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    Fei-Fei Xiong

    2013-01-01

    Full Text Available Chromosome rearrangements and fusion genes present major portion of leukemogenesis and contribute to leukemic subtypes. It is practical and helpful to detect the fusion genes in clinic diagnosis of leukemia. Present application of reverse transcription polymerase chain reaction (RT-PCR method to detect the fusion gene transcripts is effective, but time- and labor-consuming. To set up a simple and rapid system, we established a method that combined multiplex RT-PCR and microarray. We selected 15 clinically most frequently observed chromosomal rearrangements generating more than 50 fusion gene variants. Chimeric reverse primers and chimeric PCR primers containing both gene-specific and universal sequences were applied in the procedure of multiplex RT-PCR, and then the PCR products hybridized with a designed microarray. With this approach, among 200 clinic samples, 63 samples were detected to have gene rearrangements. All the detected fusion genes positive and negative were validated with RT-PCR and Sanger sequencing. Our data suggested that the RT-PCR-microarray pipeline could screen 15 partner gene pairs simultaneously at the same accuracy of the fusion gene detection with regular RT-PCR. The pipeline showed effectiveness in multiple fusion genes screening in clinic samples.

  15. Reverse transcription-polymerase chain reaction molecular testing of cytology specimens: Pre-analytic and analytic factors.

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    Bridge, Julia A

    2017-01-01

    The introduction of molecular testing into cytopathology laboratory practice has expanded the types of samples considered feasible for identifying genetic alterations that play an essential role in cancer diagnosis and treatment. Reverse transcription-polymerase chain reaction (RT-PCR), a sensitive and specific technical approach for amplifying a defined segment of RNA after it has been reverse-transcribed into its DNA complement, is commonly used in clinical practice for the identification of recurrent or tumor-specific fusion gene events. Real-time RT-PCR (quantitative RT-PCR), a technical variation, also permits the quantitation of products generated during each cycle of the polymerase chain reaction process. This review addresses qualitative and quantitative pre-analytic and analytic considerations of RT-PCR as they relate to various cytologic specimens. An understanding of these aspects of genetic testing is central to attaining optimal results in the face of the challenges that cytology specimens may present. Cancer Cytopathol 2017;125:11-19. © 2016 American Cancer Society.

  16. Multiplex Reverse Transcription-Polymerase Chain Reaction untuk Deteksi Cepat Virus Flu Burung H5N1 (MULTIPLEX REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION FOR RAPID DETECTION OF H5N1 AVIAN INFLUENZA VIRUS

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    Raden Wasito

    2015-05-01

    Full Text Available Avian influenza virus subtype H5N1 (AIV H5N1 is highly pathogenic and fatal in poultry. The virusis still endemic with low virulence rate, although it may play a critical role in causing high morbidity andmortality rates in poultry in Indonesia. In general, diagnostic approach for AIV H5N1 is based onconventional serological and viral isolation methods that have the potential to produce consumings oftime and relatively expensive cost within the laboratory without compromising test utility. Thus, amolecular approach of multiplex reverse transcription-polymerase chain reaction (mRT-PCR was developedand applied for the detection of matrix gene type A influenza viruses, AIV subtype subtype H5hemagglutinin gene with simultaneous detection of N1 nucleoprotein gene. Thirty sera specimens fromthe diseased commercial chickens that were specifically amplified positive-RT-PCR for AIV H5N1 wereselected for mRT-PCR. The mRT-PCR products were visualized by agarose gel electrophoresis and consistedof DNA fragments of AIV of 245 bp, 545 bp and 343 bp for M, H5 and N1 genes, respectively. Thus, themRT-PCR that can rapidly differentiate simultaneously between these genes is very important for thecontrol and even eradication of AIV transmission in poultry in Indonesia.

  17. Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

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    Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

    2014-09-01

    The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription

    Institute of Scientific and Technical Information of China (English)

    Stephen Johnston; Zachary Gallaher; Krzysztof Czaja

    2012-01-01

    Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2-ΔΔCt normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxin selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference β-III tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

  19. Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

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    Sudiro, T M; Zivny, J; Ishiko, H; Green, S; Vaughn, D W; Kalayanarooj, S; Nisalak, A; Norman, J E; Ennis, F A; Rothman, A L

    2001-01-01

    There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

  20. Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Screening of 29 Chromosomal Translocation in Hematologic Malignancies

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Multiplex reverse transcription-polymerase chain reaction (M-RT-PCR) has been proved to possess great clinical potential for simultaneous screening of 29 chromosomal translocations in acute leukemia. To evaluate the clinical value of M-RT-PCR in hematologic malignancies, bone marrow samples from 90 patients with various hematologic malignancies, including 25 acute myeloge nous leukemia (AML), 22 acute lymphoblastic leukemia (ALL), 27 chronic myelogenous leukemia (CML), 4 myeloproliferative diseases (MPD), 3 chronic lymphoblastic leukemia (CLL), 3 and 1 malignant histocytosis (MH) were subjected to both M-RT-PCR and chromosome karyotypic analysis. Some of cases were subjected to follow-up examination of M-RT-PCR during the period of ukemia. In our hand, 12 of 29chromosomal translocation transcripts including TEL/PDGFR, DEK/CAN, MLL/AF6, AML1/ETO,F9, BCR/ABL, MLL/MLL, PML/RARα, TLS/ERG, E2A/HLF, EVIl and HOXI1 were detected in 57 cases (63.3 %) of the 90 samples, which were in consistence with the results of karyore, M-RT-PCR had also shown good clinical relevance when used as an approach to detect minimal residual leukemia. We concluded that M-RT-PCR could be used as an effiy in the initial diagnosis of hematologic malignancies but also in subsequent monitor of minimal residual leukemia.

  1. Reverse transcription polymerase chain reaction-based method for selectively detecting vegetative cells of toxigenic Clostridium difficile.

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    Senoh, Mitsutoshi; Kato, Haru; Murase, Tomoko; Hagiya, Hideharu; Tagashira, Yasuaki; Fukuda, Tadashi; Iwaki, Masaaki; Yamamoto, Akihiko; Shibayama, Keigo

    2014-11-01

    The laboratory diagnostic methods for Clostridium difficile infection (CDI) include toxigenic culture, enzyme immunoassays (EIAs) to detect the toxins of C. difficile, and nucleic acid amplification tests (NAATs) to detect C. difficile toxin genes, but each of these methods has disadvantages; toxigenic cultures require a long time to produce results, EIAs have low sensitivity, and NAATs that target DNA cannot distinguish vegetative cells from spores and dead cells. Here we report a new detection method that uses reverse transcription polymerase chain reaction to target the toxin-gene transcripts. This method was able to specifically detect the vegetative cells of toxigenic C. difficile in fecal samples in spike tests, with a minimum detection limit of 5 × 10(2) colony-forming units per 100 mg of stool specimen. The performance of this method was also demonstrated in a pilot scale evaluation using clinical fecal specimens, which showed that this method may be more sensitive than EIA and requires a shorter time than toxigenic culture. This method could potentially be applied in the clinical laboratory to detect C. difficile in fecal specimens. The ability of this method to discriminate the presence of vegetative cells from spores and dead cells could help to further the understanding of CDI.

  2. Detection of Avian bornavirus in multiple tissues of infected psittacine birds using real-time reverse transcription polymerase chain reaction.

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    Delnatte, Pauline; Mak, Matthew; Ojkic, Davor; Raghav, Raj; DeLay, Josepha; Smith, Dale A

    2014-03-01

    Avian bornavirus (ABV), the cause of proventricular dilation disease in psittacine birds, has been detected in multiple tissues of infected birds using immunohistochemical staining (IHC) and reverse transcription polymerase chain reaction (RT-PCR). In the current study, real-time RT-PCR, using primers targeting the ABV matrix gene, was used to detect ABV in 146 tissues from 7 ABV-infected psittacine birds. Eighty-six percent of the samples tested positive, with crossing point values ranging from 13.82 to 37.82 and a mean of 22.3. These results were compared to the findings of a previous study using gel-based RT-PCR and IHC on the same samples. The agreement between the 2 RT-PCR techniques was 91%; when tests disagreed it was because samples were negative using gel-based RT-PCR but positive on real-time RT-PCR. Agreement with IHC was 77%; 16 out of 74 samples were negative using IHC but positive on real-time RT-PCR. The results suggest that real-time RT-PCR is a more sensitive technique than gel-based RT-PCR and IHC to detect ABV in tissues. The tissues that were ranked most frequently as having a high amount of viral RNA were proventriculus, kidney, colon, cerebrum, and cerebellum. Skeletal muscle, on the other hand, was found to have a consistently low amount of viral RNA.

  3. Candidate gene biodosimeters of mice and human exposure to ionizing radiation by quantitative reverse transcription polymerase chain reaction

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    Hamed Rezaeejam

    2015-01-01

    Full Text Available Understanding of cellular responses to ionizing radiation (IR is essential for the development of predictive markers useful for assessing human exposure. Biological markers of exposure to IR in human populations are of great interest for assessing normal tissue injury in radiation oncology and for biodosimetry in nuclear incidents and accidental radiation exposures. Traditional radiation exposure biomarkers based on cytogenetic assays (biodosimetry, are time-consuming and do not provide results fast enough and requires highly trained personnel for scoring. Hence, the development of rapid biodosimetry methods is one of the highest priorities. Exposure of cells to IR activates multiple signal transduction pathways, which result in complex alterations in gene-expression. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR has become the benchmark for the detection and quantification of RNA targets and is being utilized increasingly in monitoring the specific genes with more accurately and sensitively. This review evaluates the RT-qPCR as a biodosimetry method and we investigated the papers from 2000 up to now, which identified the genes-expression related the DNA repair, cell cycle checkpoint, and apoptosis induced by ionization radiation in peripheral blood and determined as biodosimeters. In conclusion, it could be say that RT-qPCR technique for determining the specific genes as biodosimeters could be a fully quantitative reliable and sensitive method. Furthermore, the results of the current review will help the researchers to recognize the most expressed genes induced by ionization radiation.

  4. A multiplex reverse transcription-polymerase chain reaction assay for Newcastle disease virus and avian pneumovirus (Colorado strain).

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    Ali, A; Reynolds, D L

    2000-01-01

    Newcastle disease virus (NDV) and avian pneumovirus (APV) cause Newcastle disease and rhinotracheitis respectively, in turkeys. Both of these viruses infect the respiratory system. A one-tube, multiplex, reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of both NDV and Colorado strain of APV (APV-Col) was developed and evaluated. The primers, specific for each virus, were designed from the matrix protein gene of APV-Col and the fusion protein gene of NDV to amplify products of 631 and 309 nucleotides, respectively. The multiplex RT-PCR assay, for detecting both viruses simultaneously, was compared with the single-virus RT-PCR assays for its sensitivity and specificity. The specific primers amplified products of predicted size from each virus in the multiplex as well as the single-virus RT-PCR assays. The multiplex RT-PCR assay was determined to be equivalent to the single-virus RT-PCR assays for detecting both NDV and APV-Col. This multiplex RT-PCR assay proved to be a sensitive method for the simultaneous and rapid detection of NDV and APV-Col. This assay has the potential for clinical diagnostic applications.

  5. DETECTION OF MICROMETASTASES OF LUNG CANCER BY USING LUNX mRNA SPECIFIC REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION

    Institute of Scientific and Technical Information of China (English)

    朱广迎; 刘德林; 王绪; 彭猛青; 刘惠; 沈万华; 张海舟; 王伟; 陈杰

    2002-01-01

    Objective: To detect of lung cancer micrometastases in peripheral blood and regional lymphatic nodes by using lunx mRNA specific reverse transcription-polymerase chain reaction (RT-PCR). Methods: RT-PCR was used to detect lunx mRNA in peripheral blood of 26 patients with lung cancer. We also detected 44 regional lymphatic nodes obtained from 25 patients with lung cancer who underwent curative lobectomy. All the 44 regional lymphatic nodes were also examined by histopathology. Micrometastatic tumor cells in the peripheral blood and regional lymphatic nodes were semiquantitatively determined with the ratio of lunx band intensity to the glyceraldehydes-3-phosphate dehydrogenase band intensity. Results: The positive detection rate of lunx mRNA in peripheral blood for non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients were 60% (12/20) and 67% (4/6) respectively. 16 (36.4%) of regional lymphatic nodes from 44 lung cancer patients were positive by RT-PCR while 6 (13.6%) were positive by histopathology (x2=6.06, P=0.014). However, no blood samples and lymphatic nodes from patients with benign pulmonary diseases or normal volunteers were positive for lunx mRNA. The positive detection rate of lunx mRNA in bone marrow of NSCLC amd SCLC patients were 65% (13/20) and 67% (4/6) respectively. Conclusion: RT-PCR amplification of lunx mRNA is an sensitive and specific means to detect early haematogenous and regional lymphatic nodes dissemination of cancer cells for patients with lung cancer.

  6. Aphids preserved in propylene glycol can be used for reverse transcription-polymerase chain reaction detection of Potato virus Y.

    Science.gov (United States)

    Nie, Xianzhou; Pelletier, Yvan; Mason, Nicola; Dilworth, Andrea; Giguère, Marie-Andrée

    2011-08-01

    The effectiveness of propylene glycol on the retention of RNA target of Potato virus Y (PVY), an aphid stylet-borne virus, in Myzus persicae was investigated in comparison to ethanol and liquid nitrogen/-80°C. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the PVY targets from the propylene glycol/ethanol/liquid nitrogen preserved single aphids after a 5min acquisition period from infected potato plants. In the liquid nitrogen/-80°C and 70% ethanol treatments, 55.6% and 38.8% aphids tested PVY-positive, respectively. In the 0-75% propylene glycol treatments, 12.2-44.7% aphids tested PVY-positive. The lowest detection rate was in the 0% (positive rate, 15.2%) and the 10% propylene glycol (positive rate, 12.2%). As the propylene glycol concentration increased to 25%, 29.8% aphids tested positive. A high PVY-positive rate was also found in 35-75% propylene glycol treatments at 44.7% (35% propylene glycol), 36.7% (50% propylene glycol) and 34.8% (75% propylene glycol), which is comparable to the rate shown in 70% ethanol. No significant difference in the positive detection rate was observed in aphids preserved in 50% propylene glycol at room temperature for 2, 4 and 10 days. These results demonstrate that propylene glycol at 25-75% can retain PVY targets effectively in aphids for an extended time period, and thus can be used in aphid traps to preserve viruliferous aphids for later RT-PCR detection of PVY.

  7. Selection of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in Brassica napus under various stress conditions.

    Science.gov (United States)

    Wang, Zheng; Chen, Yu; Fang, Hedi; Shi, Haifeng; Chen, Keping; Zhang, Zhiyan; Tan, Xiaoli

    2014-10-01

    Data normalization is essential for reliable output of quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assays, as the unsuitable choice of reference gene(s), whose expression might be influenced by exogenous treatments in plant tissues, could cause misinterpretation of results. To date, no systematic studies on reference genes have been performed in stressed Brassica napus. In this study, we investigated the expression variations of nine candidate reference genes in 40 samples of B. napus leaves subjected to various exogenous treatments. Parallel analyses by geNorm and NormFinder revealed that optimal reference genes differed across the different sets of samples. The best-ranked reference genes were PP2A and TIP41 for salt stress, TIP41 and ACT7 for heavy metal (Cr(6+)) stress, PP2A and UBC21 for drought stress, F-box and SAND for cold stress, F-box and ZNF for salicylic acid stress, TIP41, ACT7, and PP2A for methyl jasmonate stress, TIP41 and ACT7 for abscisic acid stress, and TIP41, UBC21, and PP2A for Sclerotinia sclerotiorum stress. Two newly employed reference genes, TIP41 and PP2A, showed better performances, suggesting their suitability in multiple conditions. To further validate the suitability of the reference genes, the expression patterns of BnWRKY40 and BnMKS1 were studied in parallel. This study is the first systematic analysis of reference gene selection for qRT-PCR normalization in B. napus, an agriculturally important crop, under different stress conditions. The results will contribute toward more accurate and widespread use of qRT-PCR in gene analysis of the genus Brassica.

  8. Rapid and sensitive electrochemiluminescence detection of rotavirus by magnetic primer based reverse transcription-polymerase chain reaction

    Energy Technology Data Exchange (ETDEWEB)

    Zhan Fangfang; Zhou Xiaoming [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China); Xing Da, E-mail: xingda@scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China)

    2013-01-25

    Graphical abstract: In this work, we have developed and demonstrated a magnetic primer based RT-PCR assay for ECL detection of rotavirus. In the presence of two functional primers (magnetic primer and TBR-primer) and PCR reagents, cDNA from RT was amplified directly onto MPs during PCR cycles of denaturation, annealing and extension. The resulting MPs-TBR complexes were easily loaded on the electrode surface and produced a concentrated ECL signal. The figure shows the schematic illustration of magnetic primer RT-PCR based ECL assay for rotavirus detection. Highlights: Black-Right-Pointing-Pointer A novel method for detection of rotavirus has been developed. Black-Right-Pointing-Pointer In the presence of magnetic primer, TBR-primer and PCR reagents, cDNA form RT was amplified directly onto MPs. Black-Right-Pointing-Pointer To obtain the best sensing and efficient performance, important parameters associated with the efficiency were investigated carefully. Black-Right-Pointing-Pointer The proposed method will find numerous applications in food safety field and clinical diagnosis. - Abstract: A novel method for detection of rotavirus has been developed by integrating magnetic primer based reverse transcription-polymerase chain reaction (RT-PCR) with electrochemiluminescence (ECL) detection. This is realized by accomplishing RT of rotavirus RNA in traditional way and performing PCR of the resulting cDNA fragment on the surface of magnetic particles (MPs). In order to implement PCR on MPs and achieve rapid ECL detection, forward and reverse primers are bounded to MPs and tris-(2,2 Prime -bipyridyl) ruthenium (TBR), respectively. After RT-PCR amplification, the TBR labels are directly enriched onto the surface of MPs. Then the MPs-TBR complexes can be loaded on the electrode surface and analyzed by magnetic ECL platform without any post-modification or post-incubation process. So some laborious manual operations can be avoided to achieve rapid yet sensitive detection

  9. Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Zhang, Xiaoyan; Peng, Yanmei; Wang, Ying; Zhang, Zongying; Li, Dawei; Yu, Jialin; Han, Chenggui

    2016-11-22

    Brassica yellows virus (BrYV), proposed to be a new polerovirus species, three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) have been described. This study was to develop a simple, rapid, sensitive, cost-effective method for simultaneous detection and differentiation of three genotypes of BrYV. In this study, a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection and differentiation of the three genotypes of BrYV. The three genotypes of BrYV and Tunip yellows virus (TuYV) could be differentiated simultaneously using six optimized specific oligonucleotide primers, including one universal primer for detecting BrYV, three BrYV genotype-specific primers, and a pair of primers for specific detection of TuYV. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. The mRT-PCR products were 278 bp for BrYV-A, 674 bp for BrYV-B, 505 bp for BrYV-C, and 205 bp for TuYV. Amplification of three target genotypes was optimized by increasing the PCR annealing temperatures to 62 °C. One to three fragments specific for the virus genotypes were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. No specific products could be amplified from cDNAs of other viruses which could infect crucifer crops. Detection limits of the plasmids for multiplex PCR were 100 fg for BrYV-A and BrYV-B, 10 pg for BrYV-C, and 1 pg for TuYV, respectively. The mRT-PCR was applied successfully for detection of three BrYV genotypes from field samples collected in China. The simple, rapid, sensitive, and cost-effective mRT-PCR was developed successfully for detection and differentiation of the three genotypes of BrYV.

  10. Detection and strain differentiation of infectious bronchitis virus in tracheal tissues from experimentally infected chickens by reverse transcription-polymerase chain reaction. Comparison with an immunohistochemical technique

    DEFF Research Database (Denmark)

    Handberg, Kurt; Nielsen, O.L.; Pedersen, M.W.;

    1999-01-01

    Oligonucleotide pairs were constructed for priming the amplification of fragments of nucleocapsid (N) protein and spike glycoprotein (S) genes of avian infectious bronchitis virus (IBV) by reverse transcription-polymerase chain reaction (RT-PCR). One oligonucleotide pair amplified a common segment......3896 and 793B strains of IBV, respectively, Groups of specific pathogen free chickens were experimentally inoculated with the Massachusetts (H120, M41), the D1466 and the 793B strains of IBV, and tracheal tissue preparations were made from each bird for RT-PCR and for immunohistochemistry (IHC) up to 3...

  11. Multiplex reverse transcription-polymerase chain reaction combined with on-chip electrophoresis as a rapid screening tool for candidate gene sets

    DEFF Research Database (Denmark)

    Wittig, Rainer; Salowsky, Rüdiger; Blaich, Stephanie

    2005-01-01

    Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality...... of this approach was demonstrated by low interexperimental variations of amplicon quantities after endpoint analysis. When applied to RNA samples derived from drug-sensitive and -resistant melanoma cell lines, mRT-PCR delivered results qualitatively concordant with data obtained from Northern blot and array...

  12. Rapid Amplification and Detection of Foodborne Pathogenic Rotavirus by Continuous-flow Reverse Transcription-Polymerase Chain Reaction Integrated with Online Fluorescence Analysis%集在线荧光分析的连续流动反转录-聚合酶链式反应快速扩增检测食源致病性轮状病毒

    Institute of Scientific and Technical Information of China (English)

    章春笋; 李彧媛; 王海英

    2011-01-01

    采用含RNA反转录(Reverse transcription,RT)和在线荧光分析的连续流动聚合酶链式反应(Po1ymerase chain reaction,PCR)微流控技术检测食源致病性轮状病毒.此RT-PCR微流控装置以加热铜块组成恒温带,以聚四氟乙烯毛细管微通道为反应体系构建而成.采用循环水冷却退火区,此装置能获得低至31℃的退火温度,而且温度控制及其稳定性良好,因而能满足不同PCR的要求.为了充分体现PCR微流控技术的优越性,在线荧光分析被用于检测PCR产物.当流速为7.5 mm/s时,轮状病毒RNA的cDNA扩增和在线荧光分析能在约12 min完成(扩增约10 min,在线分析约2 min).在此集成化的RT-PCR微流控装置上,Ih可完成轮状病毒RNA的RT-PCR以及其扩增产物的在线荧光分析,样品检出限达到6.4×10(4)copies/μL.%A continuous-flow polymerase chain reaction (PCR) microfluidics integrated with reverse transcription (RT) of RNA and online fluorescence analysis has been introduced, which has been successfully applied for fast amplification and identification of foodborne pathogenic Rotavirus. On this continuous-flow RT-PCR device, the isothermal heating copper blocks provides the temperatures for RT-PCR, and the polytetrafluoroethylene (PTFE) capillary was used as the RT-PCR reaction channel. By using the cycling water to cool the annealing zone, the annealing temperature as low as 31 ℃ could be obtained for the presented device, where good temperature control and stability was also achieved. Therefore, this device can meet different PCR requirements. To make the best of the advantages of microfluidic PCR, the online fluorescence analysis of amplification products has been performed. When the flow rate of 7.5 mm/s was used, the amplification of cDNA synthesized from Rotavirus RNA and then the online fluorescence analysis of amplification products could be completed in about 12 min ( about 10 min for amplification and 2 min for analysis). By using

  13. Improvement and optimization of a multiplex real-time reverse transcription polymerase chain reaction assay for the detection and typing of Vesicular stomatitis virus.

    Science.gov (United States)

    Hole, Kate; Velazquez-Salinas, Lauro; Velazques-Salinas, Lauro; Clavijo, Alfonso

    2010-05-01

    An improvement to a previously reported real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the detection of Vesicular stomatitis virus (VSV) is described. Results indicate that the new assay is capable of detecting a panel of genetically representative strains of VSV present in North, Central, and South America. The assay is specific for VSV and allows for simultaneous differentiation between Vesicular stomatitis Indiana virus and Vesicular stomatitis New Jersey virus. This real-time RT-PCR is able to detect current circulating strains of VSV and can be used for rapid diagnosis of VSV and differentiation of VSV from other vesicular diseases, such as foot-and-mouth disease.

  14. Diagnosis of enzootic pneumonia in Danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle

    DEFF Research Database (Denmark)

    Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.;

    1999-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for detection of bovine respiratory syncytial virus (BRSV) in lung tissue of naturally and experimentally infected cattle. Primers were selected from the gene coding the F fusion protein, which is relatively conserved...... among BRSV isolates. The RT-PCR assay was highly specific, it yielded positive reactions only when performed on BRSV-infected cell cultures or tissues. The detection limit of the RT-PCR assay was assessed as 5 TCID50. BRSV was detected in tissues of the respiratory tract and in the tracheobroncheal....... (7%), and Pasteurella haemolytica (7%) were the most common bacterial agents found in the lungs. BRSV was identified using a conventional antigen enzyme-linked immunosorbent assay (ELISA) in 23 (17%) animals. The established BRSV-specific RT-PCR assay yielded positive results for the same 23 animals...

  15. Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

    Science.gov (United States)

    Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L

    2009-10-01

    Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

  16. Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Li, Yan; Wu, Tao; Qi, Xian; Ge, Yiyue; Guo, Xiling; Wu, Bin; Yu, Huiyan; Zhu, Yefei; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao; Zhou, Minghao

    2013-12-01

    A novel reassortant influenza A (H7N9) virus emerged recently in China. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The sensitivity of the assay was determined to be 10 RNA copies per reaction for both HA and NA genes. No cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. One hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. The assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility.

  17. Probe-free real-time reverse transcription polymerase chain reaction assays for the detection and typing of porcine reproductive and respiratory syndrome virus in Canada.

    Science.gov (United States)

    Eschbaumer, Michael; Li, Wansi May; Wernike, Kerstin; Marshall, Frank; Czub, Markus

    2015-07-01

    Porcine reproductive and respiratory syndrome (PRRS) has tremendous impact on the pork industry in North America. The molecular diagnosis of infection with PRRS virus (PRRSV) is hampered by its considerable strain diversity. In this study, 43 previously published or newly developed primers for probe-free real-time reverse transcription polymerase chain reaction (RT-PCR) were evaluated on their sensitivity, specificity, reproducibility, and repeatability, using a diverse panel of 36 PRRSV strains as well as other arteriviruses and unrelated porcine viruses. Three primer pairs had excellent diagnostic and analytical sensitivity on par with a probe-based reference assay, absolute specificity to virus genotype and species, as well as over 95% reproducibility and repeatability across a wide dynamic range.

  18. Diagnosis of enzootic pneumonia in Danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle

    DEFF Research Database (Denmark)

    Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.

    1999-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for detection of bovine respiratory syncytial virus (BRSV) in lung tissue of naturally and experimentally infected cattle. Primers were selected from the gene coding the F fusion protein, which is relatively conserved...... among BRSV isolates. The RT-PCR assay was highly specific, it yielded positive reactions only when performed on BRSV-infected cell cultures or tissues. The detection limit of the RT-PCR assay was assessed as 5 TCID50. BRSV was detected in tissues of the respiratory tract and in the tracheobroncheal....... (7%), and Pasteurella haemolytica (7%) were the most common bacterial agents found in the lungs. BRSV was identified using a conventional antigen enzyme-linked immunosorbent assay (ELISA) in 23 (17%) animals. The established BRSV-specific RT-PCR assay yielded positive results for the same 23 animals...

  19. Comparison between specific and multiplex reverse transcription-polymerase chain reaction for detection of hepatitis A virus, poliovirus and rotavirus in experimentally seeded oysters

    Directory of Open Access Journals (Sweden)

    C Coelho

    2003-06-01

    Full Text Available Outbreaks of gastroenteritis have occurred among consumers of raw or undercooked shellfish harvested from faecally polluted waters. A multiplex reverse transcription-polymerase chain reaction (RT-PCR was applied for the simultaneous detection of hepatitis A virus (HAV, poliovirus (PV and simian rotavirus (RV-SA11 and compared with specific primers for each genome sequence. Three amplified DNA products representing HAV (192 bp, PV (394 bp and RV (278 bp were identified when positive controls were used. However, when tested on experimentally contaminated raw oysters, this method was not able to detect the three viruses simultaneously. This is probably due to the low concentration of viral RNAs present in oyster extract which were partially lost during the extracts preparation.

  20. Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

    Science.gov (United States)

    Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

    2015-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.

  1. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

    Science.gov (United States)

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

  2. Synergy between cucumber mosaic virus and zucchini yellow mosaic virus on Cucurbitaceae hosts tested by real-time reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Zeng, Rong; Liao, Qiansheng; Feng, Junli; Li, Dingjun; Chen, Jishuang

    2007-06-01

    Cucumber mosaic virus (CMV) and zucchini yellow mosaic virus (ZYMV) are two principal viruses infecting cucurbitaceous crops, and their synergy has been repeatedly observed. In our present work, a real-time reverse transcription-polymerase chain reaction procedure was established to study the accumulation kinetics of these two viruses in single and combined infections at the molecular level. The accumulations of open reading frames (ORFs) for 1a, 2a, 3a and coat protein (CP) of CMV and CP of ZYMV were tested. In the single infection, CMV-Fny ORFs accumulated to their maxima in cucumber or bottle gourd at 14 d post-inoculation (dpi), and gradually declined thereafter. ZYMV-SD CP ORF reached maximal accumulation at 14 and 28 dpi on cucumber and bottle gourd, respectively. However, when co-infected with CMV-Fny and ZYMV-SD, the maximal accumulation levels of all viral ORFs were delayed. CMV-Fny ORFs reached their maxima at 21 dpi on both hosts, and ZYMV-SDCP ORF reached maximal accumulation at 21 and 28 dpi on cucumber and bottle gourd, respectively. Generally, the accumulation levels of CMV-Fny ORFs in the co-infection were higher than those in the single infection, whereas the accumulation of ZYMV-SD CP ORF showed a reverse result.

  3. Quantitative Determination of Cucumber Mosaic Virus Genome RNAs in Virions by Real-Time Reverse Transcription-Polymerase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    Jun-Li FENG; Shao-Ning CHEN; Xiang-Shan TANG; Xian-Feng DING; Zhi-You DU; Ji-Shuang CHEN

    2006-01-01

    A real-time RT-PCR procedure using the green fluorescent dye SYBR Green I was developed for determining the absolute and relative copies of cucumber mosaic virus (CMV) genomic RNAs contained in purified virions. Primers specific to each CMV ORF were designed and selected. Sequences were then amplified with length varying from 61 to 153 bp. Using dilution series of CMV genome RNAs prepared by in vitro transcription as the standard samples, a good linear correlation was observed between their threshold cycle (Ct)values and the logarithms of the initial template amounts. The copies of genomic RNA 1, RNA 2,RNA 3 and the subgenomic RNA 4 in CMV virions were quantified by this method, and the ratios were about Our work is the first report concerning the relative amounts of different RNA fragments in CMV virions as a virus with tripartite genome.

  4. Diagnosis of Avian bornavirus infection in psittaciformes by serum antibody detection and reverse transcription polymerase chain reaction assay using feather calami.

    Science.gov (United States)

    de Kloet, Arne H; Kerski, Anelle; de Kloet, Siwo R

    2011-05-01

    Avian bornavirus (ABV) is the causative agent of proventricular dilatation disease (PDD), a highly devastating and contagious disease of psittacines (parrots and parakeets), which has resulted in the death of many captive birds. Accurate diagnosis of bornavirus infection is therefore important for the identification and isolation of infected birds. The current study showed that nonvascular contour (chest) feather calami provide a ready and minimally invasive source of RNA for the detection of ABV by reverse transcription polymerase chain reaction (RT-PCR). Storage of the feathers at room temperature for at least a month did not affect the results. Serological analysis by enzyme-linked immunosorbent assay (ELISA) showed that identification of anti-bornaviral nucleoprotein P40 antibodies can identify many birds with a past or present infection. The presence of anti-avian bornaviral P24 phosphoprotein and P16 matrix protein antibodies was quite variable, rendering these antibodies less useful for diagnosis of ABV infection. The significance of the present findings is that the use of nonvascular feathers as a source of RNA allows sample collection under conditions where storage of other samples would be difficult. Serum detection by ELISA of anti-P40 antibodies allows the identification of infected birds when RT-PCR fails. © 2011 The Author(s)

  5. Establishment and Application of a TaqMan Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Rubella Virus RNA

    Institute of Scientific and Technical Information of China (English)

    Li-Hong ZHAO; Yu-Yan MA; Hong WANG; Shu-Ping ZHAO; Wei-Ming ZHAO; Hua LI; Lei-Yi WANG

    2006-01-01

    The aim of this study was to establish and apply a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for rubella virus (RV) RNA. First, the primer and TaqMan probe concentrations, as well as reaction temperatures were optimized to establish an efficient real-time quantitative RT-PCR assay for RV RNA. Next, an RV-specific PCR amplicon was made as an external standard to estimate the linearity, amplification efficiency, analytical sensitivity and reproducibility of the real time quantitative assay. Finally, the assay was applied to quantify RVRNA in clinical samples for rubella diagnosis.The RV-specific PCR amplicon was prepared for evaluation of the assay at 503 bp, and its original concentration was 2.75×109 copies/μl. The real time quantitative assay was shown to have good linearity (R2=0.9920), high amplification efficiency (E=1.91), high sensitivity (275 copies/ml), and high reproducibility (variation coefficient range, from 1.25% to 3.58%). Compared with the gold standard, the specificity and sensitivity of the assay in clinical samples was 96.4% and 86.4%, respectively. Therefore, the established quantitative RT-PCR method is a simple, rapid, less-labored, quantitative, highly specific and sensitive assay for RV RNA.

  6. Detection of EWS-FLI1 fusion transcripts in paraffin embedded tissues of peripheral primitive neuroectodermal tumors by nested reverse transcription polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    Qixing Gong; Qinhe Fan; Zhihong Zhang; Weiming Zhang

    2005-01-01

    Objective: To assess the feasibility and significance of detecting EWS-FLIlfusion transcripts in paraffin embedded tissues of peripheral primitive neuroectodermal tumors (PNETs) by nested reverse transcription polymerase chain reaction (RT-PCR).Methods: Twelve formalin-fixed and paraffin-embedded (FFPE) samples of PNET were retrieved from archive and consultation materials,together with eight cases of controlled tumor. EWS-FLI1 fusion transcripts were detected by nested RT-PCR. Home-keeping gene β-actin was used to detect the quality of mRNA. Results: β-actin mRNA was detected in 9 of the 12 tumor cases. EWS-FLI1 fusion transcripts were detected in 6 cases, among which 4 had a "type 1" fusion transcript and 2 had a "type 2" fusion transcript. None of the controlled tumor was detected the fusion gene. Conclusion: RT-PCR is a feasible method for the detection of EWS-FLI1 fusion transcripts in FFPE tissues in PNET and the result is meaningful in differential diagnosis and prognostic evaluation.

  7. A duplex real-time reverse transcription polymerase chain reaction for the detection and quantitation of avian leukosis virus subgroups A and B.

    Science.gov (United States)

    Zhou, Gang; Cai, Wenbo; Liu, Xiaolei; Niu, Chengming; Gao, Caixia; Si, Changde; Zhang, Wei; Qu, Liandong; Han, Lingxia

    2011-05-01

    Avian leukosis is a disease that is spreading widely in the world causing large economic losses to the poultry industry. In this study, a duplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to detect and quantify avian leukosis virus subgroups A and B (ALVA/B). The assay was optimised to measure viral gp85 and chicken housekeeping (β-actin) genes. The result showed that the assay was specific for reference strains of ALVA/B subtype and no cross-reaction was detected with ALV subtypes E and J or with four other non-ALV viruses. The assay detected as few as 56 gp85 cDNA copies and was 100-fold more sensitive than a conventional RT-PCR. Seventy clinical blood samples were evaluated by both the qRT-PCR and the conventional RT-PCR assay, and the results show that 65 samples were positive by the qRT-PCR compared with 43 by the conventional RT-PCR. When this assay was used to quantify the viral load in ALV-inoculated embryos from three congenic chicken lines, the embryos from the B21 line showed the highest viral load, whereas the lowest load was found in the B5 line. This assay provides a powerful tool for quantitative detection of the ALVA/B and for the study of host genetic resistance to avian leukosis.

  8. Analytical validation of a real-time reverse transcription polymerase chain reaction test for Pan-American lineage H7 subtype Avian influenza viruses

    Science.gov (United States)

    Spackman, Erica; Ip, H.S.; Suarez, D.L.; Slemons, R.D.; Stallknecht, D.E.

    2008-01-01

    A real-time reverse transcription polymerase chain reaction test for the identification of the H7 subtype in North American Avian influenza viruses (AIVs) was first reported in 2002; however, recent AIV surveillance efforts in wild birds and H7 outbreaks in poultry demonstrated that the 2002 test did not detect all H7 AIVs present in North and South America. Therefore, a new test, the 2008 Pan-American H7 test, was developed by using recently available H7 nucleotide sequences. The analytical specificity of the new assay was characterized with an RNA panel composed of 19 H7 viruses from around the world and RNA from all hemagglutinin subtypes except H16. Specificity for North and South American lineage H7 viruses was observed. Assay limits of detection were determined to be between 103 and 104 gene copies per reaction with in vitro transcribed RNA, and 100.0 and 10 0.8 50% egg infectious doses per reaction. The 2008 Pan-American H7 test also was shown to perform similarly to the 2002 test with specimens from chickens experimentally exposed to A/Chicken/BritishColumbia/314514-2/04 H7N3 highly pathogenic AIV. Furthermore, the 2008 test was able to detect 100% (n = 27) of the H7 AIV isolates recovered from North American wild birds in a 2006-2007 sample set (none of which were detected by the 2002 H7 test).

  9. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation.

    Science.gov (United States)

    Goodell, Christa K; Zhang, Jianqiang; Strait, Erin; Harmon, Karen; Patnayak, Devi; Otterson, Tracy; Culhane, Marie; Christopher-Hennings, Jane; Clement, Travis; Leslie-Steen, Pamela; Hesse, Richard; Anderson, Joe; Skarbek, Kevin; Vincent, Amy; Kitikoon, Pravina; Swenson, Sabrina; Jenkins-Moore, Melinda; McGill, Jodi; Rauh, Rolf; Nelson, William; O'Connell, Catherine; Shah, Rohan; Wang, Chong; Main, Rodger; Zimmerman, Jeffrey J

    2016-01-01

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10(-1) to 10(-8)). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated.

  10. Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

    Science.gov (United States)

    Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

    2011-07-01

    Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

  11. A capsid gene-based real-time reverse transcription polymerase chain reaction assay for the detection of marine vesiviruses in the Caliciviridae

    Science.gov (United States)

    McClenahan, Shasta D.; Bok, Karin; Neill, John D.; Smith, Alvin W.; Rhodes, Crystal R.; Sosnovtsev, Stanislav V.; Green, Kim Y.; Romero, Carlos H.

    2009-01-01

    A real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was developed for the identification of marine vesiviruses. The primers were designed to target a 176-nucleotide fragment within a highly conserved region of the San Miguel sea lion viruses (SMSVs) capsid gene. The assay detected viral RNA from nine marine vesivirus serotypes described previously, including two serotypes (SMSV-8 and SMSV-12) not identified with presently available molecular assays, a highly-related bovine vesivirus strain (Bos-1), a mink vesivirus strain (MCV), and two novel genotypes isolated recently from Steller sea lions (SSL V810 and V1415). The real-time assay did not amplify sequences from the corresponding genomic regions of feline calicivirus (also in the genus Vesivirus) and representative members of the genus Norovirus. The rtRT-PCR assay described below may prove useful as a diagnostic tool for the detection of currently circulating, emerging and previously described marine vesiviruses in clinical samples, especially when large numbers are screened in surveillance studies of these restricted viruses. PMID:19410604

  12. Up-regulation of Cartilage Oligomeric Matrix Protein Gene Expression by Insulin-like Growth Factor-I Revealed by Real Time Reverse Transcription-Polymerase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    Hua TIAN; Ioannis STOGIANNIDIS

    2006-01-01

    Cartilage oligomeric matrix protein (COMP) strengthens cartilage by binding to type Ⅱ and typeⅨ collagen-forming bridges between collagen fibrils. It was hypothesized that perhaps one or more anabolic growth factors such as insulin-like growth factor-I (IGF-I), fibroblast growth factor-1 (FGF-1) or platelet derived growth factor-BB (PDGF-BB) increase COMP gene expression. Their effects on primary human chondrocytes and the chondrogenic cell line ATDC5 were studied using real time reverse transcript-polymerase chain reaction (RT-PCR) for quantification. IGF-I, but not the FGF-1 or PDGF-BB, up-regulated COMP gene expression by approximate 5-fold in human adult chondrocytes in a dose- and time-dependent manner. IGF-I exerted similar effects on ATDC5 cells. Results from these real time RT-PCR experiments were confirmed by transfecting into ATDC5 cells a full-length mouse COMP promoter cloned upstream of a luciferase reporter gene. On stimulation with IGF-I, the luciferase reporter activity increased by about eight times. In conclusion, IGF-I seems to be an important positive regulator of COMP, which may play an important role in an attempted repair of either traumatized or degenerated cartilage.

  13. Prostate specific membrane antigen (PSM) is expressed in various human tissues: implication for the use of PSM reverse transcription polymerase chain reaction to detect hematogenous prostate cancer spread.

    Science.gov (United States)

    Renneberg, H; Friedetzky, A; Konrad, L; Kurek, R; Weingärtner, K; Wennemuth, G; Tunn, U W; Aumüller, G

    1999-01-01

    Detection of prostate-specific membrane antigen (PSM)-mRNA expression in blood samples using reverse transcription polymerase chain reaction (RT-PCR) is discussed as a new diagnostic marker of circulating micrometastases in prostate cancer patients. We applied the RT-PCR technique to different human tissues and obtained positive signals for PSM transcripts in human genital and multiple extra-genital tissue sites. The cDNAs were prepared from different human tissues and prostatic cell lines. RT-PCR and nested RT-PCR for PSM was performed with primers derived from the published PSM cDNA. The RT-PCR fragments obtained were cloned and showed 100% sequence homology to PSM. Southern blot hybridization with labeled probes was used to confirm the specificity of the amplicons. In addition to the known PSM expression in the human brain, PSM-mRNA was detected in cDNA isolated from human testis, epididymis and seminal vesicles and in the PC-3 prostatic cancer cell line. Furthermore, we found PSM-mRNA in heart, liver, lung, kidney, spleen, and thyroid gland. The results indicate that PSM expression is not restricted to the prostate gland, but represents a more general component of genital and extra-genital human tissues. This must be considered when RT-PCR and nested RT-PCR screening for PSM expression is performed as a diagnostic measure in blood from prostate cancer patients.

  14. Development of conventional and real-time reverse transcription polymerase chain reaction assays to detect Tembusu virus in Culex tarsalis mosquitoes.

    Science.gov (United States)

    Petz, Lawrence N; Turell, Michael J; Padilla, Susana; Long, Lewis S; Reinbold-Wasson, Drew D; Smith, Darci R; O'Guinn, Monica L; Melanson, Vanessa R; Lee, John S

    2014-10-01

    Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000-fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens. © The American Society of Tropical Medicine and Hygiene.

  15. Reverse Transcription Polymerase Chain Reaction-based System for Simultaneous Detection of Multiple Lily-infecting Viruses

    Directory of Open Access Journals (Sweden)

    Ji Yeon Kwon

    2013-09-01

    Full Text Available A detection system based on a multiplex reverse transcription (RT polymerase chain reaction (PCR was developed to simultaneously identify multiple viruses in the lily plant. The most common viruses infecting lily plants are the cucumber mosaic virus (CMV, lily mottle virus (LMoV, lily symptomless virus (LSV. Leaf samples were collected at lily-cultivation facilities located in the Kangwon province of Korea and used to evaluate the detection system. Simplex and multiplex RT-PCR were performed using virus-specific primers to detect single-or mixed viral infections in lily plants. Our results demonstrate the selective detection of 3 different viruses (CMV, LMoV and LSV by using specific primers as well as the potential of simultaneously detecting 2 or 3 different viruses in lily plants with mixed infections. Three sets of primers for each target virus, and one set of internal control primers were used to evaluate the detection system for efficiency, reliability, and reproducibility.

  16. Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs.

    Directory of Open Access Journals (Sweden)

    Kelsey Haist

    Full Text Available Arenaviruses are bi-segmented, single-stranded RNA viruses that cause significant human disease. The manner in which they regulate the replication of their genome is not well-understood. This is partly due to the absence of a highly sensitive assay to measure individual species of arenavirus replicative RNAs. To overcome this obstacle, we designed a quantitative reverse transcription (RT-PCR assay for selective quantitation of each of the lymphocytic choriomeningitis virus (LCMV genomic or antigenomic RNAs. During the course of assay design, we identified a nonspecific priming phenomenon whereby, in the absence of an RT primer, cDNAs complementary to each of the LCMV replicative RNA species are generated during RT. We successfully circumvented this nonspecific priming event through the use of biotinylated primers in the RT reaction, which permitted affinity purification of primer-specific cDNAs using streptavidin-coated magnetic beads. As proof of principle, we used the assay to map the dynamics of LCMV replication at acute and persistent time points and to determine the quantities of genomic and antigenomic RNAs that are incorporated into LCMV particles. This assay can be adapted to measure total S or L segment-derived viral RNAs and therefore represents a highly sensitive diagnostic platform to screen for LCMV infection in rodent and human tissue samples and can also be used to quantify virus-cell attachment.

  17. Real-time quantitative reverse transcription-polymerase chain reaction to detect propionibacterial ribosomal RNA in the lymph nodes of Chinese patients with sarcoidosis.

    Science.gov (United States)

    Zhou, Y; Wei, Y-R; Zhang, Y; Du, S-S; Baughman, R P; Li, H-P

    2015-09-01

    The aim of this study was to investigate the diagnostic value of using the copy number of propionibacterial rRNA as a biomarker for sarcoidosis. Ribosomal RNA of Propionibacterium acnes and P. granulosum was measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) using formalin-fixed and paraffin-embedded tissue of lymph node biopsy from 65 Chinese patients with sarcoidosis, 45 with tuberculosis and 50 controls with other diseases (23 with non-specific lymphadenitis and 27 with mediastinal lymph node metastasis from lung cancer). The receiver operating characteristic (ROC) curve was analysed to determine an optimal cut-off value for diagnosis, and the diagnostic accuracy of the cut-off value was evaluated in additional tissue samples [24 patients with sarcoidosis and 22 with tuberculosis (TB)]. P. acnes or P. granulosum rRNA was detected in 48 of the 65 sarcoidosis samples but only in four of the 45 TB samples and three of the 50 control samples. Analysis of the ROC curve revealed that an optimal cut-off value of the copy number of propionibacterial rRNA for diagnosis of sarcoidosis was 50·5 copies/ml with a sensitivity and specificity of 73·8 and 92·6%, respectively. Based on the cut-off value, 19 of the 24 additional sarcoidosis samples exhibited positive P. acnes or P. granulosum, whereas only one of the 22 additional TB samples was positive, resulting in a sensitivity and specificity of 79·2 and 95·5%, respectively. These findings suggest that propionibacteria might be associated with sarcoidosis granulomatous inflammation. Detection of propionibacterial rRNA by RT-PCR might possibly distinguish sarcoidosis from TB.

  18. Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

    Science.gov (United States)

    Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

    2014-01-01

    Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

  19. A diagnostic one-step real-time reverse transcription polymerase chain reaction method for accurate detection of influenza virus type A

    Science.gov (United States)

    Behzadi, Mohammad Amin; Alborzi, Abdolvahab

    2016-01-01

    Introduction Influenza A is known as a public health concern worldwide. In this study, a novel one-step real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was designed and optimized for the detection of influenza A viruses. Material and methods The primers and probe were designed based on the analysis of 90 matrix nucleotide sequence data of influenza type A subtypes from the GenBank database of the National Center for Biotechnology Information (NCBI). The influenza virus A/Tehran/5652/2010 (H1N1 pdm09) was used as a reference. The rtRT-PCR assay was optimized, compared with that of the World Health Organization (WHO), and its analytical sensitivity, specificity and reproducibility were evaluated. In total, 64 nasopharyngeal swabs from patients with influenza-like illness (ILI) and 41 samples without ILI symptoms were tested for the virus, using conventional cell culture, direct immunofluorescence antibody (DFA) methods, and one-step rtRT-PCR with the designed primer set and probe and the WHO’s. Results The optimized assay results were similar to the WHO’s. The optimized assay results were similar to WHO’s, with non-significant differences for 10–103 copies of viral RNA/reaction (p > 0.05). It detected 10 copies of viral RNA/reaction with high reproducibility and no cross reactivity with other respiratory viruses. A specific cytopathic effect was observed in 6/64 (9.37%) of the ILI group using conventional culture and DFA staining methods; however, it was not seen in non-ILI. Also, the results of our assay and the WHO’s were similar to those of viral isolation and DFA staining. Conclusions Given the high specificity, sensitivity and reproducibility of this novel assay, it can serve as a reliable diagnostic tool for the detection of influenza A viruses in clinical specimens and lab experiments. PMID:27904520

  20. Detection of avian influenza A/H7N9/2013 virus by real-time reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Kang, Xiaoping; Wu, Weili; Zhang, Chuntao; Liu, Licheng; Feng, Huahua; Xu, Lizhi; Zheng, Xin; Yang, Honglei; Jiang, Yongqiang; Xu, Bianli; Xu, Jin; Yang, Yinhui; Chen, Weijun

    2014-09-01

    The first case of avian influenza A/H7N9 infection was reported in Shanghai in mid-February, 2013; by May 1, 2013, it had infected 127 people and caused 26 deaths in 10 provinces in China. Therefore, it is important to obtain reliable epidemiological data on the spread of this new infectious agent, a need that may be best met by the development of novel molecular methods. Here, a new method was described for the detection of avian influenza A/H7N9 using real-time reverse transcription-polymerase chain reaction (rRT-PCR). Using serial dilutions of avian influenza A H7N9 cultures, the detection limit of the assay was determined to be approximately 3.2×10(-4) HAUs (hemagglutination units) for the H7 gene and 6.4×10(-4) HAUs for N9 gene. In tests of serial dilutions of in vitro-transcribed avian influenza A H7 and N9 gene RNA, positive results were obtained for target RNA containing at least three copies of the H7 gene and six copies of the N9 gene. Thirteen throat swabs from H7N9 patients were tested; all tested positive in the assay. Specificity was evaluated by testing 18 other subtypes of influenza viruses; all tested negative. A total of 180 throat swabs from patients infected with influenza virus, including 60 from patients infected with seasonal influenza A/H1N1 virus, 60 from patients infected with pandemic influenza A/H1N1/2009 virus, 30 from patients infected with seasonal influenza A/H3N2 virus and 30 from patients infected with influenza B virus, were also tested; all tested negative.

  1. Rapid detection and differentiation of wild-type and three attenuated lapinized vaccine strains of classical swine fever virus by reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Pan, Chu-Hsiang; Jong, Ming-Hwa; Huang, Yu-Liang; Huang, Tien-Shine; Chao, Parn-Hwa; Lai, Shiow-Suey

    2008-07-01

    A simple one-step reverse transcription polymerase chain reaction (RT-PCR) method was developed based on T-rich insertions in the viral genome for simultaneous detection and differentiation of wild type and vaccine strains of Classical swine fever virus (CSFV). The CSFV-specific primers were designed to contain the sequences of the T-rich insertion sites that exist uniquely in the 3' nontranslated regions (3' NTR) of the genome of lapinized CSFV vaccine strains. By using a one-step RT-PCR or a nested PCR followed by an agarose gel electrophoresis or a multicapillary electrophoresis, the wild-type and lapinized vaccine strains of CSFV in clinical samples could be detected and accurately distinguished. These assays can be applied to at least 3 attenuated lapinized vaccine strains, lapinized Philippines Coronel (LPC), hog cholera lapinized virus (HCLV), and Chinese strain (C strain). The detection limit of the wild-type virus was 6.3 TCID(50) (50% tissue culture infective dose)/ml for RT-PCR and 0.63 TCID(50)/ml for nested PCR. In previous studies, notable T-rich insertions of 12-13 nucleotides (nt) were found in the 3' NTR of the genome of lapinized vaccine strains of CSFV. However, this study discovered that 2 T-rich insertions, 42 and 36 nt in length, are present in the viral genome of lapinized vaccine strains LPC/PRK (primary rabbit kidney) and LPC/TS (Tam-Sui), respectively. These T-rich insertions of 12, 36, and 42 nt length increases the size of PCR fragments, which are favorable genetic markers for rapid detection of and differentiation between wild-type and different lapinized vaccine strains of CSFV.

  2. Comparison of the Diagnostic Value Between Real-Time Reverse Transcription-Polymerase Chain Reaction Assay and Histopathologic Examination in Sentinel Lymph Nodes for Patients With Gastric Carcinoma.

    Science.gov (United States)

    Kwak, Yoonjin; Nam, Soo Kyung; Shin, Eun; Ahn, Sang-Hoon; Lee, Hee Eun; Park, Do Joong; Kim, Woo Ho; Kim, Hyung-Ho; Lee, Hye Seung

    2016-05-01

    Sentinel lymph node (SLN)-based diagnosis in gastric cancers has shown varied sensitivities and false-negative rates in several studies. Application of the reverse transcription-polymerase chain reaction (RT-PCR) in SLN diagnosis has recently been proposed. A total of 155 SLNs from 65 patients with cT1-2, N0 gastric cancer were examined. The histopathologic results were compared with results obtained by real-time RT-PCR for detecting molecular RNA (mRNA) of cytokeratin (CK)19, carcinoembryonic antigen (CEA), and CK20. The sensitivity and specificity of the multiple marker RT-PCR assay standardized against the results of the postoperative histological examination were 0.778 (95% confidence interval [CI], 0.577-0.914) and 0.781 (95% CI, 0.700-0.850), respectively. In comparison, the sensitivity and specificity of intraoperative diagnosis were 0.819 (95% CI, 0.619-0.937) and 1.000 (95% CI, 0.972-1.000), respectively. The positive predictive value of the multiple-marker RT-PCR assay was 0.355 (95% CI, 0.192-0.546) for predicting non-SLN metastasis, which was lower than that of intraoperative diagnosis (0.813, 95% CI, 0.544-0.960). The real-time RT-PCR assay could detect SLN metastasis in gastric cancer. However, the predictive value of the real-time RT-PCR assay was lower than that of precise histopathologic examination and did not outweigh that of our intraoperative SLN diagnosis. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

    Institute of Scientific and Technical Information of China (English)

    Zhu Yang; Guoliang Mao; Yujun Liu; Yuan-Chuan Chen; Chengjing Liu; Jun Luo; Xihan Li

    2013-01-01

    A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N 1/2009 influenza A virus.In this study,we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus.The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers.The qRT-PCR assays with the newly designed primers are highly specific,and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human,swine,and raccoon dog origin.Furthermore,the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction,respectively.When tested with 83 clinical samples,32 were detected to be positive using the qRT-PCR assays with our designed primers,while only 25 were positive by the assays with the WHO-recommended primers.These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.

  4. Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil

    Directory of Open Access Journals (Sweden)

    Marilia Farignoli Romeiro

    2016-06-01

    Full Text Available Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410 of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

  5. Development, Evaluation, and Integration of a Quantitative Reverse-Transcription Polymerase Chain Reaction Diagnostic Test for Ebola Virus on a Molecular Diagnostics Platform.

    Science.gov (United States)

    Cnops, Lieselotte; Van den Eede, Peter; Pettitt, James; Heyndrickx, Leo; De Smet, Birgit; Coppens, Sandra; Andries, Ilse; Pattery, Theresa; Van Hove, Luc; Meersseman, Geert; Van Den Herrewegen, Sari; Vergauwe, Nicolas; Thijs, Rein; Jahrling, Peter B; Nauwelaers, David; Ariën, Kevin K

    2016-10-15

     The 2013-2016 Ebola epidemic in West Africa resulted in accelerated development of rapid diagnostic tests for emergency outbreak preparedness. We describe the development and evaluation of the Idylla™ prototype Ebola virus test, a fully automated sample-to-result molecular diagnostic test for rapid detection of Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV).  The Idylla™ prototype Ebola virus test can simultaneously detect EBOV and SUDV in 200 µL of whole blood. The sample is directly added to a disposable cartridge containing all reagents for sample preparation, RNA extraction, and amplification by reverse-transcription polymerase chain reaction analysis. The performance was evaluated with a variety of sample types, including synthetic constructs and whole blood samples from healthy volunteers spiked with viral RNA, inactivated virus, and infectious virus.  The 95% limits of detection for EBOV and SUDV were 465 plaque-forming units (PFU)/mL (1010 copies/mL) and 324 PFU/mL (8204 copies/mL), respectively. In silico and in vitro analyses demonstrated 100% correct reactivity for EBOV and SUDV and no cross-reactivity with relevant pathogens. The diagnostic sensitivity was 97.4% (for EBOV) and 91.7% (for SUDV), the specificity was 100%, and the diagnostic accuracy was 95.9%.  The Idylla™ prototype Ebola virus test is a fast, safe, easy-to-use, and near-patient test that meets the performance criteria to detect EBOV in patients with suspected Ebola. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  6. Detection of infectious bursal disease virus in various lymphoid tissues of experimentally infected specific pathogen free chickens by different reverse transcription polymerase chain reaction assays

    DEFF Research Database (Denmark)

    Kabell, Susanne; Handberg, Kurt; Kusk, Mette;

    2005-01-01

    transcription polymerase chain reaction (RT-PCR) assays, including two recently developed strain-specific assays, were employed for detection of ribonucleic acid (RNA) from three different IBDV strains in bursa tissue samples from experimentally infected specific pathogen free chickens. The virus strains...

  7. Very quick reverse transcription polymerase chain reaction for detecting 2009 H1N1 influenza A using wire-guide droplet manipulationst.

    Science.gov (United States)

    You, David J; Tran, Phat L; Kwon, Hyuck-Jin; Patel, Deepa; Yoon, Jeong-Yeol

    2011-01-01

    Reverse transcription polymerase chain reaction (RT-PCR) is currently a gold standard in identifying influenza A virus, especially H1N1 flu. Typical RT-PCR assays take about 1-2 h for thermocycling, and there is a growing need to further speed up the thermocycling to less than 30 min. Additionally, the PCR assay system should be made portable as a point-of-care detection tool. There have been attempts to further speed up the PCR assays by reducing its volume. There have also been attempts to use droplet microfluidics technology to PCR, primarily to automate the PCR enrichment processes and take advantage of its small volume. In all these attempts, heating and cooling is made by conduction heat transfer. Rapid movements of droplets (immersed in oil) over three different temperature zones make very quick PCR possible, as heating/cooling will be made by convection heat transfer, whose heat transfer coefficients are much higher than that of conduction. We used our newly-invented method of wire-guide droplet manipulations towards very quick RT-PCR. Computational fluid dynamics (CFD) simulation of our system revealed that heating/cooling for each temperature change takes 1-4 s for a 10 microL droplet, as compared to >30 s in the other quick PCRs. Theoretically a 30-cycle process can take as short as 13 s x 30 cycles = 6 min 30 s. The entire system was made as a single instrument, with the components made by a milling machine and a rapid prototyping device. No additional equipment and external computers are required. With this newly developed system, 160 bp gene sequence was amplified from 2009 H1N1 influenza A (human origin). The 30-cycle process took as short as 6 min 50 s for a 10 microL droplet (with additional 4 min for reverse transcription). Its product was confirmed by traditional gel electrophoresis, subsequent imaging as well as gene sequencing, which has been very difficult with the other stationary droplet/nanodrop approaches. The proposed system has a

  8. Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method.

    Science.gov (United States)

    Zhao, Jing; Zhao, Jin-Yin; Chen, Zhi-Xia; Zhong, Wei; Li, Long-Yun; Liu, Li-Cheng; Hu, Xiao-Xu; Chen, Wei-Jun; Wang, Meng-Zhao

    2016-12-20

    Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/μl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/μl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.

  9. Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Choudhary, Nandlal; Roy, Avijit; Govindarajulu, A; Nakhla, M K; Levy, L; Brlansky, R H

    2014-09-01

    Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing. Published by Elsevier B.V.

  10. Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill

    Directory of Open Access Journals (Sweden)

    Pasquali Giancarlo

    2010-09-01

    Full Text Available Abstract Background Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR; however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings. Results By the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs indentified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. Conclusion Our study showed that expression stability varied between putative reference genes

  11. Application of real-time reverse transcription polymerase chain reaction to the detection the matrix, H5 and H7 genes of avian influenza viruses in field samples from South Korea.

    Science.gov (United States)

    Kim, Hye-Ryoung; Oem, Jae-Ku; Bae, You-Chan; Kang, Min-Su; Lee, Hee-Soo; Kwon, Yong-Kuk

    2013-03-14

    The rapid and accurate identification of the H5 and H7 subtypes of avian influenza (AI) virus is an important step for the control and eradication of highly pathogenic AI outbreaks and for the surveillance of AI viruses that have the potential to undergo changes in pathogenicity in poultry and wild birds. Currently, real-time reverse transcription polymerase chain reaction (RRT-PCR) is routinely used for the rapid detection of the H5 and H7 genes, but misidentification is frequent for emergent isolates and viruses isolated from diverse regions due to the high sequence variation among AI viruses. In this study, an RRT-PCR method was tested for the detection of matrix, H5 and H7 genes from diverse subtypes of AI viruses and from field samples obtained through AI surveillance in South Korea over the last four years. Both RRT-PCR and conventional experiment (virus isolation using egg inoculation followed by reverse transcription polymerase chain reaction) agreed on the virus-positive samples. And the comparison of the results with 174 clinical samples showed a high level of agreement without decreasing the specificity and sensitivity. This assay could be useful tool for the rapid detection of AI using the field samples from domestic poultry and wild birds in South Korea, and continuous regional updates is needed to validate primer sets as the AI virus evolves.

  12. Primary A (H1N1) pdm09 Influenza Pneumonia Diagnosed on Reverse Transcription-polymerase Chain Reaction (RT-PCR) of Bronchoalveolar Lavage Fluid but not Rapid Tests with Nasopharyngeal Swabs.

    Science.gov (United States)

    Ohkura, Noriyuki; Tani, Mayuko; Nishitsuji, Masaru; Nishi, Koichi

    2015-01-01

    A 47-year-old man with a fever was highly suspected of having influenza A infection since his wife and son who lived with him had been diagnosed with influenza A. Although repeated rapid tests with a nasopharyngeal swab showed negative findings, the patient developed bilateral pneumonia and reverse transcription polymerase chain reaction (PCR) for A (H1N1) pdm09 virus in the bronchoalveolar lavage fluid was positive. We therefore diagnosed him with primary influenza pneumonia and initiated treatment with peramivir plus corticosteroids, which rapidly improved his condition. During the influenza season, sample collection from the lower airway and PCR should be considered for the definitive diagnosis of primary influenza viral pneumonia.

  13. Classical swine fever virus detection: results of a real-time reverse transcription polymerase chain reaction ring trial conducted in the framework of the European network of excellence for epizootic disease diagnosis and control

    DEFF Research Database (Denmark)

    Hoffmann, Bernd; Blome, Sandra; Bonilauri, Paolo

    2011-01-01

    The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time RT......-PCR protocols and a standardized protocol commonly used by the Friedrich-Loeffler-Institute (FLI) on a panel of well-characterized samples. In general, all participants produced results within the acceptable range. The FLI assay, several in-house assays, and the commercial kits had high analytical sensitivity...... and specificity values. Nevertheless, some in-house systems had unspecific reactions or suboptimal sensitivity with only a single CSFV genotype. Follow-up actions involved either improvement of suboptimal assays or replacement of specific laboratory assays with the FLI protocol, with or without modifications...

  14. Significance of detecting circulating hepatocellular carcinoma cells in peripheral blood of hepatocellular carcinoma patients by nested reverse transcription-polymerase chain reaction and its clinical value: a retrospective study.

    Science.gov (United States)

    Liu, Yang; Wang, Yue-ru; Wang, Long; Song, Rui-mei; Zhou, Bo; Song, Zhen-shun

    2014-01-01

    Circulating hepatocellular carcinoma cells may be detected by reverse transcription-polymerase chain reaction. We investigated the relationship between circulating hepatocellular carcinoma cells and hepatoma patient survival after different managements and survival periods. Peripheral vein blood (5 ml) samples were obtained from 113 patients with hepatocellular carcinoma and from 33 control subjects (9 with liver cirrhosis after hepatitis B, 14 with chronic hepatitis B, 10 healthy individuals) between January 1, 2009, and December 31, 2013. To detect circulating hepatocellular carcinoma cells in peripheral blood, alpha-fetoprotein messenger RNA was amplified from total RNA extracted from whole blood by reverse transcription-polymerase chain reaction. Alpha-fetoprotein messenger RNA was detected in 59 blood samples from the hepatocellular carcinoma patients (59/113, 52.2%). In contrast, there were no clinical control subjects whose samples showed detectable alpha-fetoprotein messenger RNA. The presence of alpha-fetoprotein messenger RNA in blood seemed to be correlated with the stage (by TNM classification) of hepatocellular carcinoma, serum alpha-fetoprotein value, and the presence of intrahepatic metastasis, portal vein thrombosis, tumor diameter and/or distant metastasis. In addition, alpha-fetoprotein messenger RNA was detected in the blood of 25 patients showing distant metastasis at extrahepatic organs (100%), in contrast to 32 of 88 cases without metastasis (36.4%). All the patients with hepatocellular carcinoma were followed. Seventeen patients with resection of a T 2 stage hepatocellular carcinoma had a survival of 3.2 years after surgical management, 38 cases with resection of a T3 stage hepatocellular carcinoma had a 1.3-year survival, and only 37 cases with T4 stage disease after different treatments except surgery survived for 0.6 years (P <0.01). The presence of alpha-fetoprotein messenger RNA in peripheral blood may be an indicator of circulating

  15. Porcine reproductive and respiratory syndrome virus: Interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods

    DEFF Research Database (Denmark)

    Wernike, Kerstin; Bonilauri, Paolo; Dauber, Malte

    2012-01-01

    To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American...... (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays...

  16. Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription-polymerase chain reaction (RT-PCR) method.

    Science.gov (United States)

    Figueiredo, L T; Batista, W C; Igarashi, A

    1997-01-01

    We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 microliters assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37 degrees C for reverse transcription followed by 30 cycles of two-step PCR amplification (92 degrees C for 60 seconds, 53 degrees C for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10(3, 6) TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination.

  17. Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR method

    Directory of Open Access Journals (Sweden)

    FIGUEIREDO Luiz Tadeu Moraes

    1997-01-01

    Full Text Available We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

  18. Differentiation of canine distemper virus isolates in fur animals from various vaccine strains by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations in china

    Directory of Open Access Journals (Sweden)

    Zhao Jianjun

    2011-02-01

    Full Text Available Abstract In order to effectively identify the vaccine and field strains of Canine distemper virus (CDV, a new differential diagnostic test has been developed based on reverse transcription-polymerase chain reaction (RT-PCR and restriction fragment length polymorphism (RFLP. We selected an 829 bp fragment of the nucleoprotein (N gene of CDV. By RFLP analysis using BamHI, field isolates were distinguishable from the vaccine strains. Two fragments were obtained from the vaccine strains by RT-PCR-RFLP analysis while three were observed in the field strains. An 829 nucleotide region of the CDV N gene was analyzed in 19 CDV field strains isolated from minks, raccoon dogs and foxes in China between 2005 and 2007. The results suggest this method is precise, accurate and efficient. It was also determined that three different genotypes exist in CDV field strains in fur animal herds of the north of China, most of which belong to Asian type. Mutated field strains, JSY06-R1, JSY06-R2 and JDH07-F1 also exist in Northern China, but are most closely related to the standard virulent strain A75/17, designated in Arctic and America-2 genetype in the present study, respectively.

  19. The use of a one-step real-time reverse transcription polymerase chain reaction (rRT-PCR) for the surveillance of viral hemorrhagic septicemia virus (VHSV) in Minnesota.

    Science.gov (United States)

    Phelps, Nicholas B D; Patnayak, Devi P; Jiang, Yin; Goyal, Sagar M

    2012-12-01

    Viral hemorrhagic septicemia virus (VHSV) is a highly contagious and pathogenic virus of fish. The virus infects more than 70 fish species worldwide, in both fresh and salt water. A new viral strain (VHSV-IVb) has proven both virulent and persistent, spreading throughout the Great Lakes of North America and to inland water bodies in the region. To better understand the geographic distribution of the virus, we used a modified real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for high-throughput testing of fish for VHSV. The assay was shown to be twice as sensitive as the gold standard, virus isolation, and did not cross react with other viruses found in fish. In addition, the diagnostic turnaround time was reduced from 28 to 30 d for virus isolation to 2-4 d for rRT-PCR. To demonstrate the usefulness of the rRT-PCR assay, 115 high-priority water bodies in Minnesota were tested by both methods from April 2010 to June 2011. All survey sites tested negative for VHSV by both methods. The survey results have informed fisheries managers on the absence of VHSV in Minnesota and have better prepared them for the eventual arrival of the disease. In addition, the results demonstrate the value of this rRT-PCR as a surveillance tool to rapidly identify an outbreak so that it can be controlled in a timely manner.

  20. Reverse transcription polymerase chain reaction (RT-PCR based detection and serotyping of FMD Virus from field samples of Gazipur, Bangladesh, and adaptation of the virus in BHK-21 cell

    Directory of Open Access Journals (Sweden)

    Mohammad Ashraful Alam

    2015-09-01

    Full Text Available The study aimed for the detection and serotyping of Foot and Mouth Disease virus (FMDV circulating in Kapasia Upazila, Gazipur district of Bangladesh during 2013. Twelve samples comprising of tongue epithelium (n=8 and inter digital tissue (n=4 were collected from suspected cattle, and inocula were prepared. The inocula were inoculated into confluent BHK-21 cell line for virus propagation. After 3 subsequent passages; progressive cytopathic effects (CPE specific for FMDV i.e., rounding and flattening of cells, breaking down of the intercellular bridge and finally cell death (almost 100% were observed; these were indicative of successful virus propagation in the cells. Viral RNA was extracted, and Reverse Transcription Polymerase Chain Reaction (RT-PCR was performed using three sets of primers corresponding to the serotype and lsquo;O', and lsquo;Asia-1' and and lsquo;A', respectively. Out of the 12 samples, 10 (83.33% were found to be positive for FMDV, and all of those were of serotype and lsquo;O'. It is concluded that FMDV serotype and lsquo;O' is circulating among the cattle of Gazipur district, Bangladesh. [J Adv Vet Anim Res 2015; 2(3.000: 291-295

  1. Field detection of eastern equine encephalitis virus in the Amazon Basin region of Peru using reverse transcription-polymerase chain reaction adapted for field identification of arthropod-borne pathogens.

    Science.gov (United States)

    O'Guinn, Monica L; Lee, John S; Kondig, John P; Fernandez, Roberto; Carbajal, Faustino

    2004-02-01

    In support of efforts to develop rapid diagnostic assays for use in the field, reverse transcription-polymerase chain reaction (RT-PCR) assays were developed to detect arboviruses circulating in the Amazon Basin region of Peru. Previous knowledge of arthropod/pathogen relationships allowed a focused evaluation to be conducted in November 2000 that assessed the feasibility and reliability of a mobile, rapid, field-expedient RT-PCR diagnostic system aimed at detecting eastern equine encephalitis virus (EEEV) in Culex (Melanoconion) pedroi mosquitoes. Modifications were made to a commercially available mobile molecular laboratory kit and assay procedures were tailored for use under harsh environmental conditions with field-collected and field-processed mosquitoes. From CO2 baited mosquito light traps, 3,227 Cx. (Mel.) pedroi mosquitoes were collected and sorted into 117 pools. The pools were processed and assayed in the field by RT-PCR and five of those pools were found positive for EEEV. Laboratory sequence analysis confirmed the presence of two distinct subtypes of EEEV.

  2. Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory.

    Science.gov (United States)

    Bao, Jian R; Huard, Thomas K; Piscitelli, Arelis E; Tummala, Praveena R; Aleemi, Virginia E; Coon, Stephanie L; Master, Ronald N; Lewinski, Michael A; Clark, Richard B

    2011-12-01

    Pandemic 2009 H1N1 is normally susceptible to oseltamivir, but variants harboring the H275Y (CAC → TAC) mutation exhibit resistance. We describe the use of a combined reverse-transcription polymerase chain reaction (RT-PCR)/pyrosequencing approach to identify the H275 residue. A total of 223 specimens were tested with this method: 216 randomly selected clinical specimens positive for 2009 H1N1 and 7 cell-culture supernatants from the Centers for Disease Control and Prevention (CDC; 4 resistant, 3 susceptible 2009 H1N1 strains). The assay detected H275Y in 1 clinical respiratory sample (0.5%) and all 4 oseltamivir-resistant strains from the CDC; the remaining 215 clinical and 3 susceptible CDC specimens were wild-type. Sanger sequencing confirmed the results for 50 of 50 selected isolates. The RT-PCR/pyrosequencing method was highly specific, producing no amplicons or valid sequences from samples containing non-H1N1 viruses or bacteria. Our findings suggest that this method provides a rapid tool for H275Y detection, with high sensitivity and potential benefit for patient care.

  3. Broadly reactive pan-paramyxovirus reverse transcription polymerase chain reaction and sequence analysis for the detection of Canine distemper virus in a case of canine meningoencephalitis of unknown etiology

    Science.gov (United States)

    Schatzberg, Scott J.; Li, Qiang; Porter, Brian F.; Barber, Renee M.; Claiborne, Mary Kate; Levine, Jonathan M.; Levine, Gwendolyn J.; Israel, Sarah K.; Young, Benjamin D.; Kiupel, Matti; Greene, Craig; Ruone, Susan; Anderson, Larry; Tong, Suxiang

    2016-01-01

    Despite the immunologic protection associated with routine vaccination protocols, Canine distemper virus (CDV) remains an important pathogen of dogs. Antemortem diagnosis of systemic CDV infection may be made by reverse transcription polymerase chain reaction (RT-PCR) and/or immunohistochemical testing for CDV antigen; central nervous system infection often requires postmortem confirmation via histopathology and immunohistochemistry. An 8-month-old intact male French Bulldog previously vaccinated for CDV presented with multifocal neurologic signs. Based on clinical and postmortem findings, the dog’s disease was categorized as a meningoencephalitis of unknown etiology. Broadly reactive, pan-paramyxovirus RT-PCR using consensus-degenerate hybrid oligonucleotide primers, combined with sequence analysis, identified CDV amplicons in the dog’s brain. Immunohistochemistry confirmed the presence of CDV antigens, and a specific CDV RT-PCR based on the phosphoprotein gene identified a wild-type versus vaccinal virus strain. This case illustrates the utility of broadly reactive PCR and sequence analysis for the identification of pathogens in diseases with unknown etiology. PMID:19901287

  4. Genotypic characterization of Indian isolates of infectious bursal disease virus strains by reverse transcription-polymerase chain reaction combined with restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Priyadharsini, C V; Senthilkumar, T M A; Raja, P; Kumanan, K

    2016-03-01

    The reverse transcription PCR (RT-PCR) combined with restriction fragment length polymorphism (RFLP) is used for the differentiation of classical virulent (cv), virulent (v) and very virulent (vv) strains of infectious bursal disease virus (IBDV) isolates from chicken bursal tissues in southern states of India. In the present study, six different isolates (MB11, HY12, PY12, BGE14, VCN14 and NKL14) of IBDV strains were subjected for genotyping along with vaccine virus (Georgia, intermediate strain) using RT-PCR for amplification of a 743 bp sequence in the hypervariable region of VP2 gene followed by restriction enzyme digestion with 5 different restriction enzymes (BspMI, SacI, HhaI, StuI and SspI). The RT-PCR products obtained from vvIBDV strains were digested by SspI enzyme except PY12, BGE14 and MB11 isolates. The SacI digested the isolate MB11, PY12 and the vaccine strain, but it did not cleave the very virulent isolates of IBDV. HhaI cleaved all the isolates with different restriction profile patterns. StuI digested all the vvIBDV isolates and BspMI was not able to differentiate field isolates from vaccine strain. Though RT-PCR combined with RFLP is a genotypic method, further confirmation of serotypes to distinguish the vvIBDV from cvIBDV has to be carried out using pathogenicity studies.

  5. Porcine reproductive and respiratory syndrome virus: interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods.

    Science.gov (United States)

    Wernike, Kerstin; Bonilauri, Paolo; Dauber, Malte; Errington, Jane; LeBlanc, Neil; Revilla-Fernández, Sandra; Hjulsager, Charlotte; Isaksson, Mats; Stadejek, Tomasz; Beer, Martin; Hoffmann, Bernd

    2012-09-01

    To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.

  6. Lanthanide chelate complementation and hydrolysis enhanced luminescent chelate in real-time reverse transcription polymerase chain reaction assays for KLK3 transcripts.

    Science.gov (United States)

    Alinezhad, Saeid; Väänänen, Riina-Minna; Lehmusvuori, Ari; Karhunen, Ulla; Soukka, Tero; Kähkönen, Esa; Taimen, Pekka; Alanen, Kalle; Pettersson, Kim

    2014-01-01

    The requirement for high-performance reporter probes in real-time detection of polymerase chain reaction (PCR) has led to the use of time-resolved fluorometry of lanthanide chelates. The aim of this study was to investigate the applicability of the principle of lanthanide chelate complementation (LCC) in comparison with a method based on hydrolysis enhancement and quenching of intact probes. A real-time reverse transcription (RT) PCR assay for kallikrein-related peptidase 3 (KLK3, model analyte) was developed by using the LCC detection method. Both detection methods were tested with a standard series of purified PCR products, 20 prostatic tissues, 20 healthy and prostate cancer patient blood samples, and female blood samples spiked with LNCaP cells. The same limit of detection was obtained with both methods, and two cycles earlier detection with the LCC method was observed. KLK3 messenger RNA (mRNA) was detected in all tissue samples and in 1 of 20 blood samples identically with both methods. The background was 30 times lower, and the signal-to-background (S/B) ratio was 3 times higher, when compared with the reference method. Use of the new reporter method provided similar sensitivity and specificity as the reference method. The lower background, the improved S/B ratio, and the possibility of melting curve analysis and single nucleotide polymorphism (SNP) detection could be advantages for this new reporter probe.

  7. Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis.

    Science.gov (United States)

    Köster, Natascha; Schmiermund, Alexandra; Grubelnig, Stefan; Leber, Jasmin; Ehlicke, Franziska; Czermak, Peter; Salzig, Denise

    2016-06-01

    For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A260 values (representing up to 40.8 μg RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A260/A280 and A260/A230 ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low Ct values of ∼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering.

  8. Comparative Diagnosis of Human Bocavirus 1 Respiratory Infection With Messenger RNA Reverse-Transcription Polymerase Chain Reaction (PCR), DNA Quantitative PCR, and Serology.

    Science.gov (United States)

    Xu, Man; Arku, Benedict; Jartti, Tuomas; Koskinen, Janne; Peltola, Ville; Hedman, Klaus; Söderlund-Venermo, Maria

    2017-05-15

    Human bocavirus (HBoV) 1 can cause life-threatening respiratory tract infection in children. Diagnosing acute HBoV1 infection is challenging owing to long-term airway persistence. We assessed whether messenger RNA (mRNA) detection would correlate better than DNA detection with acute HBoV1 infection. Paired serum samples from 121 children with acute wheezing were analyzed by means of serology. Quantitative polymerase chain reaction (PCR) and reverse-transcription (RT) PCR were applied to nasopharyngeal swab (NPS) samples from all acutely HBoV1-infected children and from controls with nonacute infection. By serology, 16 of 121 children (13.2%) had acute HBoV1 infection, all of whom had HBoV1 DNA in NPS samples, and 12 of 16 (75%) had HBoV1 mRNA. Among 25 children with nondiagnostic results, 6 had HBoV1 DNA in NPS samples, and 1 had mRNA. All 13 mRNA-positive samples exhibited high DNA loads (≥106 copies/mL). No mRNA persisted for 2 weeks, whereas HBoV1 DNA persisted for 2 months in 4 children; 1 year later all 15 samples were DNA negative. Compared with serology, DNA PCR had high clinical sensitivity (100%) but, because of viral persistence, low specificity (76%). In contrast, mRNA RT-PCR had low clinical sensitivity (75%) but high specificity (96%). A combination of HBoV1 serology and nasopharyngeal DNA quantitative PCR and mRNA RT-PCR should be used for accurate diagnosis of HBoV1 infection.

  9. Detection of Viral Hemorrhagic Septicemia Virus by Quantitative Reverse Transcription Polymerase Chain Reaction from Two Fish Species at Two Sites in Lake Superior

    Science.gov (United States)

    Cornwell, Emily R.; Eckerlin, Geofrey E.; Getchell, Rodman G.; Groocock, Geoffrey H.; Thompson, Tarin M.; Batts, William N.; Casey, Rufina N.; Kurath, Gael; Winton, James R.; Bowser, Paul R.; Bain, Mark B.; Casey, James W.

    2011-01-01

    Viral hemorrhagic septicemia virus (VHSV) was first detected in the Laurentian Great Lakes in 2005 during a mortality event in the Bay of Quinte, Lake Ontario. Subsequent analysis of archived samples determined that the first known isolation of VHSV in the Laurentian Great Lakes was from a muskellunge Esox masquinongy collected in Lake St. Clair in 2003. By the end of 2008, mortality events and viral isolations had occurred in all of the Laurentian Great Lakes except Lake Superior. In 2009, a focused disease surveillance program was designed to determine whether VHSV was also present in Lake Superior. In this survey, 874 fish from 7 sites along the U.S. shoreline of Lake Superior were collected during June 2009. Collections were focused on nearshore species known to be susceptible to VHSV. All fish were dissected individually by using aseptic techniques and were tested for the presence of VHSV genetic material by use of a quantitative reverse transcription (qRT) polymerase chain reaction (PCR) targeting the viral nucleoprotein gene. Seventeen fish from two host species at two different sites tested positive at low levels for VHSV. All attempts to isolate virus in cell culture were unsuccessful. However, the presence of viral RNA was confirmed independently in five fish by using a nested PCR that targeted the glycoprotein (G) gene. Partial G gene sequences obtained from three fish were identical to the corresponding sequence from the original 2003 VHSV isolate (MI03) from muskellunge. These detections represent the earliest evidence for the presence of VHSV in Lake Superior and illustrate the utility of the highly sensitive qRT-PCR assay for disease surveillance in aquatic animals.

  10. Evaluation and application of a one-step duplex real-time reverse transcription polymerase chain reaction assay for the rapid detection of influenza A (H7N9) virus from poultry samples.

    Science.gov (United States)

    Bao, Hongmei; Ma, Yong; Shi, Jianzhong; Zeng, Xianying; Zhao, Yuhui; Wang, Xiurong; Chen, Hualan

    2015-10-01

    In China, a novel reassortant influenza A (H7N9) virus, which has caused 435 cases of human infection, has recently emerged. Most cases of human infections with the H7N9 virus are known to be associated with a poultry farm and live-poultry markets. In this study, a one-step duplex real-time reverse transcription polymerase chain reaction (RRT-PCR) assay was developed for the simultaneous detection of the hemagglutinin (HA) and neuraminidase (NA) genes of the H7N9 virus for effective surveillance and early diagnosis of cases from clinical samples collected from live-poultry markets or poultry farms. The detection limit of this assay was as low as 0.1 EID50 of H7N9 viruses, which is similar to the detection limit of the real-time RT-PCR assay released by the Word Health Organization. The coefficients of variation (CVs) of both inter-assay and intra-assay reproducibility were less than 1.55 %, showing good reproducibility. No cross-reactivity was observed with RNA of other subtypes of influenza virus or other avian respiratory viruses. The assay can effectively detect H7N9 influenza virus RNA from multiple sources, including chickens, pigeons, ducks, humans, and the environment. Furthermore, the RRT-PCR assay was evaluated with more than 700 clinical samples collected from live-poultry markets and 120 experimentally infected chicken samples. Together, these results indicate that the duplex RRT-PCR assay is a specific, sensitive, and efficient diagnostic method for the epidemiological surveillance and diagnosis of H7N9 virus from different sources, particularly poultry samples.

  11. Development and validation of a real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay for investigation of wild poliovirus type 1-South Asian (SOAS) strain reintroduced into Israel, 2013 to 2014.

    Science.gov (United States)

    Hindiyeh, M Y; Moran-Gilad, J; Manor, Y; Ram, D; Shulman, L M; Sofer, D; Mendelson, E

    2014-02-20

    In February 2013, wild poliovirus type 1 (WPV1) was reintroduced into southern Israel and resulted in continuous silent circulation in the highly immune population. As a part of the public health emergency response, a novel real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed, to allow for the sensitive and specific detection of the circulatingWPV1-South Asian (SOAS) strain. Specific primers and probes derived from the VP-1 region were designed, based on sequenced sewage isolates, and used to simultaneously amplify this WPV1-SOAS sequence together with bacteriophage MS-2 as internal control. High titre WPV1-SOAS stock virus was used for assay optimisation and 50 processed sewage samples collected from southern Israel and tested by reference culture based methods were used for analytical validation of the assay’s performance. The limit of detection of the multiplex qRT-PCR (SOAS/MS-2) assay was 0.1 plaque-forming unit (pfu)/reaction (20 pfu/mL) for WPV1-SOAS RNA with 100% sensitivity, specificity, positive and negative predictive values when compared to the culture based method. The turnaround time was rapid, providing results for environmental samples within 24 to 48 hours from completion of sewage processing, instead of five to seven days by culture-based analysis. Direct sewage testing by qRT-PCR assay proved to be a useful tool for rapid detection and environmental surveillance of WPV1-SOAS circulating strain during emergency response. Application of the approach for detection of WPV1-SOAS in stool samples obtained during acute flaccid paralysis (AFP) surveillance or field surveys should be further evaluated.

  12. Sensitivity and specificity of real-time reverse transcription polymerase chain reaction, histopathology, and immunohistochemical labeling for the detection of Rift Valley fever virus in naturally infected cattle and sheep.

    Science.gov (United States)

    Odendaal, Lieza; Fosgate, Geoffrey T; Romito, Marco; Coetzer, Jacobus A W; Clift, Sarah J

    2014-01-01

    Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), histopathology, and immunohistochemical labeling (IHC) were performed on liver specimens from 380 naturally infected cattle and sheep necropsied during the 2010 Rift Valley fever (RVF) epidemic in South Africa. Sensitivity (Se) and specificity (Sp) of real-time RT-PCR, histopathology, and IHC were estimated in a latent-class model using a Bayesian framework. The Se and Sp of real-time RT-PCR were estimated as 97.4% (95% confidence interval [CI] = 95.2-98.8%) and 71.7% (95% CI = 65-77.9%) respectively. The Se and Sp of histopathology were estimated as 94.6% (95% CI = 91-97.2%) and 92.3% (95% CI = 87.6-95.8%), respectively. The Se and Sp of IHC were estimated as 97.6% (95% CI = 93.9-99.8%) and 99.4% (95% CI = 96.9-100%), respectively. Decreased Sp of real-time RT-PCR was ascribed to cross-contamination of samples. Stratified analysis of the data suggested variations in test accuracy with fetuses and severely autolyzed specimens. The Sp of histopathology in fetuses (83%) was 9.3% lower than the sample population (92.3%). The Se of IHC decreased from 97.6% to 81.5% in the presence of severe autolysis. The diagnostic Se and Sp of histopathology was higher than expected, confirming the value of routine postmortem examinations and histopathology of liver specimens. Aborted fetuses, however, should be screened using a variety of tests in areas endemic for RVF, and results from severely autolyzed specimens should be interpreted with caution. The most feasible testing option for countries lacking suitably equipped laboratories seems to be routine histology in combination with IHC.

  13. Fluorescence dynamics of green fluorescent protein in AOT reversed micelles

    NARCIS (Netherlands)

    Uskova, M.A.; Borst, J.W.; Hink, M.A.; Hoek, van A.; Schots, A.; Klyachko, N.L.; Visser, A.J.W.G.

    2000-01-01

    We have used the enhanced green fluorescent protein (EGFP) to investigate the properties of surfactant-entrapped water pools in organic solvents (reversed micelles) with steady-state and time-resolved fluorescence methods. The surfactant used was sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and the

  14. Prompt detection of influenza A and B viruses using the BD Veritor™ System Flu A+B, Quidel® Sofia® Influenza A+B FIA, and Alere BinaxNOW® Influenza A&B compared to real-time reverse transcription-polymerase chain reaction (RT-PCR).

    Science.gov (United States)

    Dunn, Jim; Obuekwe, Joy; Baun, Traci; Rogers, Justin; Patel, Twinkle; Snow, Linda

    2014-05-01

    The performance characteristics of rapid influenza diagnostic tests vary widely. This study evaluated the BD Veritor™ System Flu A+B (Veritor; BD Diagnostics, Sparks, MD, USA), Quidel® Sofia® Influenza A+B FIA (Sofia; Quidel Corp., San Diego, CA, USA), and Alere BinaxNOW® Influenza A&B (Binax; Alere Scarborough, Inc., Scarborough, ME, USA) compared to reverse transcription-polymerase chain reaction (RT-PCR) for detection of influenza viruses in nasal wash specimens from 240 pediatric patients. Positive percent agreements for influenza A and B virus detection were 93.8% and 94.2%, 95.8% and 98.1%, and 79.2% and 80.8% for Veritor, Sofia, and Binax, respectively. The Veritor and Binax tests demonstrated negative percent agreements >97.9% for detection of both influenza viruses, but the negative percent agreement of the Sofia test was 91.1% for influenza A and 70.7% for influenza B virus. Overall, the Veritor and Sofia tests were nearly as sensitive as RT-PCR and considerably more sensitive than Binax for detection of influenza viruses. However, the accuracy of the Sofia test was significantly lower than either Veritor or Binax.

  15. The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

    Science.gov (United States)

    Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John

    2017-07-01

    Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  16. 逆转录-聚合酶链反应检测滑膜肉瘤石蜡包埋组织中SYT-SSX融合基因%Detection of SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    魏永昆; 王坚; 朱雄增; 施达仁; 久(冈)正典; 桥本洋

    2002-01-01

    目的探讨用逆转录-聚合酶链反应(RT-PCR)技术检测滑膜肉瘤石蜡包埋组织中SYT-SSX融合基因的可行性。方法 我们采用RT-PCR方法对37例福尔马林固定、石蜡包埋的滑膜肉瘤组织中SYT-SSX融合基因转录本进行了检测。为探讨SYT-SSX融合基因对滑膜肉瘤的特异性,一系列非滑膜肉瘤的肿瘤标本作为阴性对照。融合基因检测结果用测序的方法进行了证实。结果 37例滑膜肉瘤中33例(89.2%)可检测出SYT-SSX融合基因。34例非滑膜肉瘤的肿瘤标本中均未显示SYT-SSX融合基因产物扩增信号。此34例标本中均可检测到PBGD mRNA表达。33例SYT-SSX阳性滑膜肉瘤中,SYT-SSX1阳性22例,SYT-SSX2阳性6例,其余5例无法区分融合基因类型。融合基因类型与组织学亚型间存在相关性。所有10例双相型滑膜肉瘤均为SYT-SSX1型,而所有SYT-SSX2阳性滑膜肉瘤均为单相型(P<0.05)。 结论 我们的结果提示,RT-PCR技术可以用于存档的福尔马林固定、石蜡包埋组织,作为滑膜肉瘤诊断和鉴别诊断敏感而且可靠的技术。SYT-SSX融合基因类型与组织学亚型间存在相关性。SYT-SSX2型仅见于单相型滑膜肉瘤。%Objective To assess the feasibility of detecting SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction (RT-PCR).Methods RT-PCR was used to amplify the SYT-SSX fusion transcripts using archival formalin-fixed paraffin-embedded tumor specimens from a series of 37 synovial sarcoma cases. To investigate the specificity of the SYT-SSX fusion transcripts, a variety of non-synovial sarcoma tumors were included in the study as negative controls. The detected messages derived from fusion genes were confirmed by subsequent sequence analysis. Results SYT-SSX fusion transcripts were detected in 33 of 37 (89.2%) synovial sarcomas. None of the 34 cases of non

  17. Application of Reverse Transcription-Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Method to Identify H1 Genotype of Measles Virus Infection and Measles Vaccine Related Cases%应用限制性片段长度多态性分析方法鉴别H1基因型麻疹野病毒感染病例和麻疹疫苗相关病例

    Institute of Scientific and Technical Information of China (English)

    李立群; 余文; 赵智娴; 丁峥嵘

    2012-01-01

    Objective Application the method to rapid identify the wild measles strain and China measles vaccine strain in Yunnan province. Method To apply method of reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) and sequence analysis method to detect 8 strains of measles virus. Results The RT-PCR-RFLP method for H1 genotype was used for identifying 8 strains of measles virus, the 5 specimens of wild-type, measles viruses RT-PCR products were cut into 2 fragments, 408 bp and 159 bp, the results showed that 5 specimens of wild-type measles viruses were caused by H1 genotype. The RT-PCR-RFLP method for China vaccine strain identification was used for identifying 3 specimen of measles. 1 specimen RT-PCR products were cut into 2 fragments, 287 bp and 151 bp, which showed that this case was caused by China vaccine strain. 2 specimens of measles were non-H1 wild-type measles viruses. Conclusion The RT-PCR-RFLP method can be used to identify H1 genotype of measles virus and measles vaccine strain. The results are same as the method of nucleoprotein gene sequencing.%目的 在云南省应用H1基因型麻疹野病毒鉴定方法,鉴别H1基因型麻疹野病毒感染病例,应用中国麻疹疫苗株病毒鉴定方法,鉴别接种麻疹疫苗后出现的麻疹样病例是否为麻疹疫苗相关病例.方法 采用逆转录-聚合酶链反应-限制性片段长度多态性分析(Reverse Transcription-Polymerase Chain Reaction-Restriction Fragment Length Polymorphism,RT-PCR-RFLP)方法、序列分析方法鉴别8株麻疹病毒.结果 应用H1基因型麻疹野病毒RT-PCR-RFLP方法对8份标本进行鉴定,其中5份标本RT-PCR产物经 Sal I酶切后,被切成两个片段,分别为408个碱基对(base pair,bp)和159bp,显示5个病例是由H1基因型麻疹野病毒引起的.应用中国麻疹疫苗株病毒RT-PCR-RFLP方法对3份标本进行鉴定,其中1份标本RT-PCR产物经Af1Ⅱ酶切,被切成两个片段,分别为287

  18. Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET

    NARCIS (Netherlands)

    Subach, F.V.; Zhang, L.; Gadella, T.W.J.; Gurskaya, N.G.; Lukyanov, K.A.; Verkhusha, V.V.

    2010-01-01

    We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF stat

  19. Graphene as a Reversible and Spectrally Selective Fluorescence Quencher

    Science.gov (United States)

    Salihoglu, Omer; Kakenov, Nurbek; Balci, Osman; Balci, Sinan; Kocabas, Coskun

    2016-09-01

    We report reversible and spectrally selective fluorescence quenching of quantum dots (QDs) placed in close proximity to graphene. Controlling interband electronic transitions of graphene via electrostatic gating greatly modifies the fluorescence lifetime and intensity of nearby QDs via blocking of the nonradiative energy transfer between QDs and graphene. Using ionic liquid (IL) based electrolyte gating, we are able to control Fermi energy of graphene in the order of 1 eV, which yields electrically controllable fluorescence quenching of QDs in the visible spectrum. Indeed, our technique enables us to perform voltage controllable spectral selectivity among quantum dots at different emission wavelengths. We anticipate that our technique will provide tunable light-matter interaction and energy transfer that could yield hybrid QDs-graphene based optoelectronic devices with novel functionalities, and additionally, may be useful as a spectroscopic ruler, for example, in bioimaging and biomolecular sensing. We propose that graphene can be used as an electrically tunable and wavelength selective fluorescence quencher.

  20. Reversible thermochromic polymer film embedded with fluorescent organogel nanofibers.

    Science.gov (United States)

    Kim, Hyungwoo; Chang, Ji Young

    2014-11-18

    We report a reversible thermochromic nanocomposite polymer film composed of fluorescent organogel fibers and a highly cross-linked polymer matrix. A series of cyano-substituted oligo(p-phenylenevinylene) (CN-OPV) derivatives were synthesized by the reaction of dialdehydes with phenyl or naphthyl acetonitrile under basic conditions. Among the CN-OPV derivatives, NA-DBA having naphtyl moieties and dodecyloxy chains formed a stable organogel in a cross-linkable monomeric solvent (ethylene glycol dimethacrylate). The organogel showed a thermoreversible sol-gel transition, accompanying the emission color change. A nanocomposite polymer film obtained by photopolymerization of the organogel between two quartz plates also exhibited reversible thermochromism. Under 365 nm irradiation, the orange color of the film at 25 °C became yellowish green at 120 °C. The fluorescence spectroscopy, DSC, and microscopy results determined that the thermally reversible self-assembly of NA-DBA occurred in the polymer matrix, resulting in reversible thermochromism. The melted gelator molecules at 120 °C did not diffuse into the polymer matrix probably because of poor interactions of the gelator molecules with the polymer matrix. The NA-DBA molecules dispersed in poly(methyl methacrylate), without forming a supramolecular structure, did not show thermochromism.

  1. Graphene as a Reversible and Spectrally Selective Fluorescence Quencher

    Science.gov (United States)

    Salihoglu, Omer; Kakenov, Nurbek; Balci, Osman; Balci, Sinan; Kocabas, Coskun

    2016-01-01

    We report reversible and spectrally selective fluorescence quenching of quantum dots (QDs) placed in close proximity to graphene. Controlling interband electronic transitions of graphene via electrostatic gating greatly modifies the fluorescence lifetime and intensity of nearby QDs via blocking of the nonradiative energy transfer between QDs and graphene. Using ionic liquid (IL) based electrolyte gating, we are able to control Fermi energy of graphene in the order of 1 eV, which yields electrically controllable fluorescence quenching of QDs in the visible spectrum. Indeed, our technique enables us to perform voltage controllable spectral selectivity among quantum dots at different emission wavelengths. We anticipate that our technique will provide tunable light-matter interaction and energy transfer that could yield hybrid QDs-graphene based optoelectronic devices with novel functionalities, and additionally, may be useful as a spectroscopic ruler, for example, in bioimaging and biomolecular sensing. We propose that graphene can be used as an electrically tunable and wavelength selective fluorescence quencher. PMID:27652976

  2. A halochromic stimuli-responsive reversible fluorescence switching 3, 4, 9, 10-perylene tetracarboxylic acid dye for fabricating rewritable platform

    Science.gov (United States)

    Hariharan, P. S.; Pitchaimani, J.; Madhu, Vedichi; Anthony, Savarimuthu Philip

    2017-02-01

    3, 4, 9, 10-perylene tetracarboxylic acid (PTCA), a strongly fluorescent water soluble dye with halochromic functionality showed pH dependent reversible fluorescence switching. The strong fluorescence of PTCA (Φf = 0.67) in basic medium was completely quenched upon acidification. The fluorescent PTCA has been transferred on to a solid substrate (filter paper and glass plate) that also showed reversible off-on fluorescence switching by acid/base and drying/water vapor exposure. The reversible fluorescence switching of PTCA could be of potential interest for fabricating rewritable fluorescent medium.

  3. Modulation of a solid-state reversible fluorescent photoswitching based on a controllable photochromic pyrazolones

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Hu; Guo, Jixi [Key Laboratory of Material and Technology for Clean Energy, Ministry of Education, Key Laboratory of Advanced Functional Materials, Autonomous Region, Institute of Applied Chemistry, Xinjiang University, Urumqi 830046, Xinjiang (China); Jia, Dianzeng, E-mail: jdz@xju.edu.cn [Key Laboratory of Material and Technology for Clean Energy, Ministry of Education, Key Laboratory of Advanced Functional Materials, Autonomous Region, Institute of Applied Chemistry, Xinjiang University, Urumqi 830046, Xinjiang (China); Guo, Mingxi; Le, Fuhe; Liu, Lang; Wu, Dongling [Key Laboratory of Material and Technology for Clean Energy, Ministry of Education, Key Laboratory of Advanced Functional Materials, Autonomous Region, Institute of Applied Chemistry, Xinjiang University, Urumqi 830046, Xinjiang (China); Li, Feng [State Laboratory of Surface and Interface Science and Technology, School of Materials and Chemical Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, Henan (China); University of Texas, M. D. Anderson Cancer Center, Houston, TX 77002 (United States)

    2014-08-15

    A novel solid-state reversible fluorescence photoswitching system (FPS) based on photochromism of photochromic pyrazolones has been developed by employing phosphor Sr{sub 2}P{sub 2}O{sub 7} co-doped with europium ion and chlorine ion (Sr{sub 2}P{sub 2}O{sub 7}–EC) and 1,3-diphenyl-4-(3-chlorobenzal)-5-hydroxypyrazole-4-phenylsemicarbazone (1a) as the fluorescence dye and the photochromic compound, respectively. With carefully selected components, the absorption band of the keto-form photochromic pyrazolones well overlaps with the emission peak of Sr{sub 2}P{sub 2}O{sub 7}–EC. The fluorescence emission intensity of Sr{sub 2}P{sub 2}O{sub 7}–EC is efficiently modulated by the photoisomerization of 1a with controlling the exposure time in the solid state. The fluorescence photoswitching system displayed high fluorescence quenching efficiency and remarkable fatigue resistance. It can be repeated 7 cycles without observable the changes of emission intensity. A fluorescence quenching efficiency can be achieved with a reversible colour change from white to yellow. - Graphical abstract: A novel fluorescence photoswitching system based on doping inorganic fluorescence dye into photochromic pyrazolones was constructed successfully. Its fluorescence emission could be efficiently modulated by the photoisomerization of pyrazolones. - Highlights: • A solid-state fluorescence photoswitching material was prepared. • Photoswitching is due to energy transfer between pyrazolone and fluorescence dye. • It exhibits excellent fluorescence contrast and fatigue resistance in the solid state.

  4. Guest-responsive reversible swelling and enhanced fluorescence in a super-absorbent, dynamic microporous polymer.

    Science.gov (United States)

    Rao, K Venkata; Mohapatra, Sudip; Maji, Tapas Kumar; George, Subi J

    2012-04-10

    A swell idea! The guest-responsive reversible swelling and fluorescence enhancement of a dynamic, microporous polymer network is presented. Guest-induced breathing of hydrophobic pores imparts multi-functional properties, such as super-absorbency, phase-selective swelling of oil from water and encapsulation of C(60) (see figure), to this soft micro-porous organic polymer.

  5. Flexible self-supporting nanofibers thin films showing reversible photochromic fluorescence.

    Science.gov (United States)

    Gao, Rui; Cao, Ding; Guan, Yan; Yan, Dongpeng

    2015-05-13

    Highly sensitive stimuli-responsive fluorescent films play an important role in smart sensors and readable optical devices. However, systems involving light-driven fluorescence changes are still limited compared with photochromic materials that simply change color upon photostimulation. Herein, by incorporation of stilbene-based molecules into a poly(vinyl alcohol) host, we have developed new flexible self-supporting nanofiber films that exhibited fast and obvious photochromic fluorescence (PCF). The reversible transfer between two fluorescent states can be easily recycled. Fluorescence microscopy and atomic force microscopy images supplied in situ evidence of changes in fluorescence and surface morphology, respectively. Density functional theoretical calculations showed that the PCF can be attributed to photoisomerization of the stilbene-based molecules. Therefore, based on the combination of experimental and theoretical studies, this work not only supplies new stilbene-based systems with light-induced fluorescence change, but also gives detailed understanding on the photoisomerization and PCF processes of the nanofibers systems. We anticipate that these PCF films can be applied in erasable memory devices and antiforgery materials, and that our strategy may be extended to other systems to fabricate multistimuli-responsive fluorescent materials.

  6. Early Detection and Serotyping of Dengue Viruses Clinical Isolates Using Reverse Transcription Polymerase Chain Reaction (RT-PCR 2 Primers

    Directory of Open Access Journals (Sweden)

    Abdul Rahman Siregar

    2015-11-01

    Full Text Available Recently several methods for confirming Dengue Virus have been developed involve virus isolation, detection of virus antigen, and nucleic acid using PCR. It has been reported that rapid detection method for confirming DHF by Multiplex RT-PCR had been successfully developed. It was more effective than the other methods with a high sensitivity and specivicity were 100% at the early phase (day 1-3. This study was designed to develop rapid detection and serotyping methods for Dengue Virus using RT-PCR 2 primers (Dcon and preM with annealing temperature was 57oC. The whole blood samples were collected from suspected dengue fever patients that had been confirmed with NS1 kit from R.S. Persahabatan DKI Jakarta and R.S. Prof. Dr. Sardjito DI Yogyakarta during Februari-August 2009. The PCR products showed that in 12 samples, 100 % were postitive with different pattern among the serotypes especially for DEN1 and DEN2, but not for DEN3 and Den4.  This method was also able to confirm the double infection DEN2-DEN3, but not for the other ones because of the unspecific pattern. From the results, it indicated that the 2 primers can be a promising early detection and serotyping method of Dengue Virus which infected the DHF patients. Key words: Dengue Virus, DHF, early detection, serotyping, RT-PCR 2 primers.

  7. Reference Genes for Gene Expression Analysis by Real-time Reverse Transcription Polymerase Chain Reaction of Renal Cell Carcinoma

    DEFF Research Database (Denmark)

    Bjerregaard, Henriette; Pedersen, Shona; Kristensen, Søren Risom

    2011-01-01

    Differentiation between malignant renal cell carcinoma and benign oncocytoma is of great importance to choose the optimal treatment. Accurate preoperative diagnosis of renal tumor is therefore crucial; however, existing imaging techniques and histologic examinations are incapable of providing...

  8. An Immunofluorescence-assisted Microfluidic Single Cell Quantitative Reverse Transcription Polymerase Chain Reaction Analysis of Tumour Cells Separated from Blood

    Directory of Open Access Journals (Sweden)

    Kazunori Hoshino

    2015-11-01

    matched the results from a few thousand cells. Some markers (e.g., ER, HER2 that are commonly used for cancer identification showed relatively large deviations in expres‐ sion levels. However, others (e.g., GRB7 showed devia‐ tions that are small enough to supplement single cell disease profiling.

  9. Detection of Zaire Ebola virus by real-time reverse transcription-polymerase chain reaction, Sierra Leone, 2014.

    Science.gov (United States)

    Liu, Licheng; Sun, Yang; Kargbo, Brima; Zhang, Chuntao; Feng, Huahua; Lu, Huijun; Liu, Wenseng; Wang, Chengyu; Hu, Yi; Deng, Yongqiang; Jiang, Jiafu; Kang, Xiaoping; Yang, Honglei; Jiang, Yongqiang; Yang, Yinhui; Kargbo, David; Qian, Jun; Chen, Weijun

    2015-09-15

    During the 2014 Ebola virus disease (EVD) outbreak, a real-time quantitative polymerase chain reaction was established to detect and identify the Zaire Ebola virus. We describe the use of this assay to screen 315 clinical samples from EVD suspected person in Sierra Leone. The detection rate in blood samples was 77.81% (207/266), and there were relatively higher detection rate (79.32% and 81.42%, respectively) during the first two weeks after onset of symptoms. In the two weeks that followed, the detection rate declined to 66.67% and 25.00%, respectively. There was the highest virus load at the first week and then decreased. The detection rate in swab samples was 89.79% (44/49). This may be benefit from the included patients. 46 of 49 swab samples were collected from died patients. Taken together, the results presented here indicate that the assay specifically and sensitively detects Zaire Ebola virus.

  10. Field Evaluation of a Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Vesicular Stomatitis Virus

    Science.gov (United States)

    Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the US livestock industries because VS is an OIE reportable disease and also clinically mimics foot-and-mouth disease. Rapid and accurate differentiation of these two diseases is critical ...

  11. National Policy for solid waste and reverse logistics of fluorescent lamps after consumption: a case study

    Directory of Open Access Journals (Sweden)

    William Cestari

    2016-03-01

    Full Text Available Fluorescent lamps are used increasingly today. Considered energy-saving lamps, they become hazardous waste at the end of their life cycle. They are composed partly of mercury, considered harmful to human health. Fluorescent lamps, together with tires and pesticide containers, fall under federal legislation that establishes and regulates their proper disposal. In 2010, Federal Law 12.305 / 2010 was created, establishing the National Policy on Solid Waste (PNRS, making businesses and consumers responsible for the proper disposal of their waste. This article aims to analyze the reverse logistics process of fluorescent lamps post-consumption, in a public education institution. The research used case studies and interviews with managers of the materials administration and warehouse departments of the institution. It was identified that since 2010, the educational institution has not been performing reverse logistics and has been accumulating hazardous material in its warehouses. This paper contributes a suggestion to improve the reverse logistics process and worker safety, and to prevent environmental risks.

  12. Reverse-tone ultraviolet nanoimprint lithography with fluorescent UV-curable resins

    Science.gov (United States)

    Uehara, Takuya; Kubo, Shoichi; Nakagawa, Masaru

    2015-06-01

    The usefulness of fluorescent ultraviolet (UV)-curable resins for reverse-tone UV nanoimprint lithography was demonstrated. Nanoimprinted concave patterns were fabricated as an underlayer on a Au-deposited substrate using a fluorescent UV-curable resin with a low etching durability after curing. The underlayer was coated with another fluorescent UV-curable resin with a high etching durability after curing. Fluorescence microscopic observation allowed the inspection of the morphological uniformity and thickness of both under and top layers in a nondestructive manner. UV-curing of the top layer in combination with pressing with a flat substrate was necessary for obtaining a flattened top layer surface, which was responsible for allowing the reverse-tone UV nanoimprint lithography. We demonstrated the fabrication of 10-nm-thick Au split-ring resonator structures with a line width of 55 ± 4 nm corresponding to the underlayer concave resist patterns by dry etching during Ar ion milling under the condition that the etching rate ratio of the underlayer to the top layer was 6.

  13. Primary Role of the Chromophore Bond Length Alternation in Reversible Photoconversion of Red Fluorescence Proteins

    Science.gov (United States)

    Drobizhev, Mikhail; Hughes, Thomas E.; Stepanenko, Yuriy; Wnuk, Pawel; O'Donnell, Kieran; Scott, J. Nathan; Callis, Patrik R.; Mikhaylov, Alexander; Dokken, Leslie; Rebane, Aleksander

    2012-01-01

    Rapid photobleaching of fluorescent proteins can limit their use in imaging applications. The underlying kinetics is multi-exponential and strongly depends on the local chromophore environment. The first, reversible, step may be attributed to a rotation around one of the two exocyclic C-C bonds bridging phenol and imidazolinone groups in the chromophore. However it is not clear how the protein environment controls this motion - either by steric hindrances or by modulating the electronic structure of the chromophore through electrostatic interactions. Here we study the first step of the photobleaching kinetics in 13 red fluorescent proteins (RFPs) with different chromophore environment and show that the associated rate strongly correlates with the bond length alternation (BLA) of the two bridge bonds. The sign of the BLA appears to determine which rotation is activated. Our results present experimental evidence for the dominance of electronic effects in the conformational dynamics of the RFP chromophore. PMID:23008753

  14. Primary Role of the Chromophore Bond Length Alternation in Reversible Photoconversion of Red Fluorescence Proteins

    Science.gov (United States)

    Drobizhev, Mikhail; Hughes, Thomas E.; Stepanenko, Yuriy; Wnuk, Pawel; O'Donnell, Kieran; Scott, J. Nathan; Callis, Patrik R.; Mikhaylov, Alexander; Dokken, Leslie; Rebane, Aleksander

    2012-09-01

    Rapid photobleaching of fluorescent proteins can limit their use in imaging applications. The underlying kinetics is multi-exponential and strongly depends on the local chromophore environment. The first, reversible, step may be attributed to a rotation around one of the two exocyclic C-C bonds bridging phenol and imidazolinone groups in the chromophore. However it is not clear how the protein environment controls this motion - either by steric hindrances or by modulating the electronic structure of the chromophore through electrostatic interactions. Here we study the first step of the photobleaching kinetics in 13 red fluorescent proteins (RFPs) with different chromophore environment and show that the associated rate strongly correlates with the bond length alternation (BLA) of the two bridge bonds. The sign of the BLA appears to determine which rotation is activated. Our results present experimental evidence for the dominance of electronic effects in the conformational dynamics of the RFP chromophore.

  15. Highly selective and reversible chemosensor for Pd(2+) detected by fluorescence, colorimetry, and test paper.

    Science.gov (United States)

    Wang, Mian; Liu, Xiaomei; Lu, Huizhe; Wang, Hongmei; Qin, Zhaohai

    2015-01-21

    A "turn-on" fluorescent and colorimetric chemosensor (RBS) for Pd(2+) has been designed and synthesized through introduction of sulfur as a ligand atom to Rhodamine B. RBS exhibits high selectivity (freedom from the interference of Hg(2+ )in particular) and sensitivity toward Pd(2+) with a detection limit as low as 2.4 nM. RBS is also a reversible sensor, and it can be made into test paper to detect Pd(2+) in pure water. Compared to the chemosensors that introduced phosphorus to Rhodamine to detect Pd(2+), RBS can be synthesized more simply and economically.

  16. Multi-ion detection and molecular switching behaviour of reversible dual fluorescent sensor

    Science.gov (United States)

    Basheer, Sabeel M.; Muralisankar, M.; Anjana, T. V.; Aneesrahman, K. N.; Sreekanth, Anandaram

    2017-07-01

    The selective chemosensing behaviour of imidazole bisthiocarbohydrazone (IBTC) towards F- and Cu2 + are studied via colorimetric, UV-Visible, fluorescence spectra studies, and binding constants were calculated. The 1H NMR titration study strongly support that the deprotonation of IBTC followed by the hydrogen bond formation via N1sbnd H1 and N2sbnd H2 protons with fluoride ion. The fluorescence inactive IBTC-Cu complex became fluorescence active in the presence of perchlorate (ClO4-) ion. The selective detection of perchlorate ion was also explained. The F- sensing mechanism of IBTC has been investigated by Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TDDFT) methods. The theoretical outcomes well reproduce the experimental results. And it concluded the Nsbnd H protons, nearby the imine group was first captured by the added F- ion and then deprotonation happened followed by the formation of hydrogen bond. The IBTC found good reversibility character with the alternative addition of Ca2 + and F-. The multi-ion detection of IBTC was used to construct the NOR, OR and INHIBITION molecular logic gates.

  17. Fluorescence via Reverse Intersystem Crossing from Higher Triplet States in a Bisanthracene Derivative

    CERN Document Server

    Sato, Tohru; Haruta, Naoki; Pu, Yong-Jin

    2016-01-01

    To elucidate the high external quantum efficiency observed for organic light-emitting diodes using a bisanthracene derivative, BD1, as the emitting molecule, off-diagonal vibronic coupling constants (VCCs) between the excited states of BD1, which govern non-radiative transition rates, were calculated employing time-dependent density functional theory. The VCCs were analysed based on the concept of vibronic coupling density. The VCC calculations suggest a fluorescence via higher triplets (FvHT) mechanism, which entails the conversion of a T$_4$ exciton generated during electrical excitation into an S$_2$ exciton via reverse intersystem crossing (RISC); moreover, the S$_2$ exciton relaxes to a fluorescent S$_1$ exciton because of large vibronic coupling between S$_2$ and S$_1$. This mechanism is valid as long as the relaxation of triplet states higher than T$_1$ to lower states is suppressed. The symmetry-controlled thermally activated delayed fluorescence (SC-TADF) and inverted singlet and triplet (iST) struct...

  18. Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield

    Science.gov (United States)

    Chaturvedi, Sumit K.; Zhao, Huaying; Schuck, Peter

    2017-04-01

    Sedimentation velocity analytical ultracentrifugation with fluorescence detection has emerged as a powerful method for the study of interacting systems of macromolecules. It combines picomolar sensitivity with high hydrodynamic resolution, and can be carried out with photoswitchable fluorophores for multi-component discrimination, to determine the stoichiometry, affinity, and shape of macromolecular complexes with dissociation equilibrium constants from picomolar to micromolar. A popular approach for data interpretation is the determination of the binding affinity by isotherms of weight-average sedimentation coefficients, sw. A prevailing dogma in sedimentation analysis is that the weight-average sedimentation coefficient from the transport method corresponds to the signal- and population-weighted average of all species. We show that this does not always hold true for systems that exhibit significant signal changes with complex formation - properties that may be readily encountered in practice, e.g., from a change in fluorescence quantum yield. Coupled transport in the reaction boundary of rapidly reversible systems can make significant contributions to the observed migration in a way that cannot be accounted for in the standard population-based average. Effective particle theory provides a simple physical picture for the reaction-coupled migration process. On this basis we develop a more general binding model that converges to the well-known form of sw with constant signals, but can account simultaneously for hydrodynamic co-transport in the presence of changes in fluorescence quantum yield. We believe this will be useful when studying interacting systems exhibiting fluorescence quenching, enhancement or Forster resonance energy transfer with transport methods.

  19. Single-tube fluorescent product-enhanced reverse transcriptase assay with Ampliwax (STF-PERT) for retrovirus quantitation.

    Science.gov (United States)

    Sears, Johnna F; Khan, Arifa S

    2003-03-01

    A TaqMan fluorescent probe-based product enhanced reverse transcriptase (RT) assay is described in which the RT and polymerase chain reaction (PCR) steps are set-up in a single tube, in two compartments separated by Ampliwax (designated as single-tube fluorescent product-enhanced reverse transcriptase assay (STF-PERT)). This simplification of the two-step method resulted in increased assay reproducibility and handling efficiency while maintaining the sensitivity of the PERT assay (PERT assay can be used to quantitate low amounts of retrovirus in clinical and research materials and to evaluate retrovirus contamination in cell substrates and biological products in human use.

  20. A Turn-on and Reversible Fluorescence Sensor for Zinc Ion Based on 4,5-Diazafluorene Schiff Base.

    Science.gov (United States)

    Li, Hui; Zhang, ShuJiang; Gong, ChenLiang; Wang, JianZhi; Wang, Feng

    2016-09-01

    A new 4,5-diazafluorene-based fluorescent chemosensor has been synthesized by Schiff base condensation of 9,9-bis(3,5-dimethyl-4-aminophenyl)-4,5-diazafluorene with salicylaldehyde. The interaction of Schiff base with different metal ions has been studied over photofluorescent spectra. The results showed that Schiff base exhibited 194-fold enhancements in fluorescence at 465 nm after Zn(2+) ions. Such fluorescent responses could be detected by naked eye under UV-lamp. The complex solution (L-Zn(2+)) exhibited reversibility with EDTA.

  1. A reversible DNA-silver nanoclusters-based molecular fluorescence switch and its use for logic gate operation.

    Science.gov (United States)

    Huang, Zhenzhen; Ren, Jinsong; Qu, Xiaogang

    2012-03-01

    Molecule-like silver nanoclusters (AgNCs) with few to tens of atoms are highly sensitive to the sequence and structure of DNA stabilizers. In this paper, a novel pH-triggered reversible molecular fluorescence switch is developed by taking advantage of the DNA-dependent fluorescence pH response of AgNCs. The DNA-AgNCs fluorescence switch simultaneously addresses concerns of simple construction strategy, efficient design and organic-solvent-free operation. Moreover, the excellent photostability and biocompatibility of AgNCs provide great potential for application of the DNA-AgNCs fluorescence switch in the development of functional molecular devices. Specifically, we apply the DNA-AgNCs fluorescence switch combined with the DNA sequence-dependent pH response pattern of AgNCs for construction of molecular logic gates.

  2. Simulation of Far-Field Superresolution Fluorescence Imaging with Two-Color One-Photon Excitation of Reversible Photoactivatable Protein

    Institute of Scientific and Technical Information of China (English)

    WANG Chen; QIAO Ling-Ling; MAO Zheng-Le

    2011-01-01

    We propose to achieve far-field super-resolution imaging by using offset two-color one-photon (2C1P) excitation of reversible photoactivatable fluorescence proteins. Due to the distinctive photoswitching performance of the proteins, such as dronpa, the fluorescence emission will only come from the overlapped region of activation beam and excitation beam. The analysis solution of rate equation shows that the resolution of offset 2C1P microscope is "engineered" by laser power of excitation and activation beams and the power ratio between them. Superior lateral and transverse resolution is theoretically demonstrated compared with conventional fluorescence scanning microscopy.%@@ We propose to achieve far-field super-resolution imaging by using offset two-color one-photon(2C1P) excitation of reversible photoactivatable fluorescence proteins.Due to the distinctive photoswitching performance of the proteins,such as dronpa,the fluorescence emission will only come from the overlapped region of activation beam and excitation beam.The analysis solution of rate equation shows that the resolution of offset 2C1P microscope is "engineered" by laser power of excitation and activation beams and the power ratio between them.Superior lateral and transverse resolution is theoretically demonstrated compared with conventional fluorescence scanning microscopy.

  3. Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity

    Science.gov (United States)

    Shen, Duanwen; Bai, Mingfeng; Tang, Rui; Xu, Baogang; Ju, Xiaoming; Pestell, Richard G.; Achilefu, Samuel

    2013-04-01

    Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

  4. Investigating the allosteric reverse signalling of PARP inhibitors with microsecond molecular dynamic simulations and fluorescence anisotropy.

    Science.gov (United States)

    Marchand, Jean-Rémy; Carotti, Andrea; Passeri, Daniela; Filipponi, Paolo; Liscio, Paride; Camaioni, Emidio; Pellicciari, Roberto; Gioiello, Antimo; Macchiarulo, Antonio

    2014-10-01

    The inhibition of the poly(ADP-ribose) polymerase (PARP) family members is a strategy pursued for the development of novel therapeutic agents in a range of diseases, including stroke, cardiac ischemia, cancer, inflammation and diabetes. Even though some PARP-1 inhibitors have advanced to clinical setting for cancer therapy, a great deal of attention is being devoted to understand the polypharmacology of current PARP inhibitors. Besides blocking the catalytic activity, recent works have shown that some PARP inhibitors exhibit a poisoning activity, by trapping the enzyme at damaged sites of DNA and forming cytotoxic complexes. In this study we have used microsecond molecular dynamics to study the allosteric reverse signalling that is at the basis of such an effect. We show that Olaparib, but not Veliparib and HYDAMTIQ, is able to induce a specific conformational drift of the WGR domain of PARP-1, which stabilizes PARP-1/DNA complex through the locking of several salt bridge interactions. Fluorescence anisotropy assays support such a mechanism, providing the first experimental evidence that HYDAMTIQ, a potent PARP inhibitor with neuroprotective properties, is less potent than Olaparib to trap PARP-1/DNA complex.

  5. Highly sensitive and fast-responsive fluorescent chemosensor for palladium: reversible sensing and visible recovery.

    Science.gov (United States)

    Li, Honglin; Fan, Jiangli; Hu, Mingming; Cheng, Guanghui; Zhou, Danhong; Wu, Tong; Song, Fengling; Sun, Shiguo; Duan, Chunying; Peng, Xiaojun

    2012-09-24

    The well-known rhodamine spiro-lactam framework offers an ideal model for the development of fluorescence-enhanced chemosensors through simple and convenient syntheses. Herein, we report a new tridentate PNO receptor, which was introduced into a rhodamine spiro-lactam system to develop Pd(2+)-chemosensor RPd4, that displayed significantly improved sensing properties for palladium. Compound RPd4 shows a very fast response time (about 5 s), high sensitivity (5 nM), and excellent specificity for Pd(2+) ions over other PGE ions (Pt(2+), Rh(3+), and Ru(3+)). In addition, RPd4 displays quite different responses to different valence states of the Pd ions, that is, very fast response towards Pd(2+) ions but slow response towards Pd(0), which may provide us with a convenient method for the selective discrimination of Pd species in different valence states. According to proof-of-concept experiments, RPd4 has potential applications in Pd(2+)-analysis in drug compounds, water, soil, and leaf samples. Owing to its good reversibility, RPd4 can also be used as a sensor material for the selective detection and visual recovery of trace Pd(2+) ions in environmental samples.

  6. Structuration in the Interface of Direct and Reversed Micelles of Sucrose Esters, Studied by Fluorescent Techniques

    Science.gov (United States)

    Sandoval, Catalina; Ortega, Anakenna; Sanchez, Susana A.; Morales, Javier; Gunther, German

    2015-01-01

    Background Reactors found in nature can be described as micro-heterogeneous systems, where media involved in each micro-environment can behave in a markedly different way compared with the properties of the bulk solution. The presence of water molecules in micro-organized assemblies is of paramount importance for many chemical processes, ranging from biology to environmental science. Self-organized molecular assembled systems are frequently used to study dynamics of water molecules because are the simplest models mimicking biological membranes. The hydrogen bonds between sucrose and water molecules are described to be stronger (or more extensive) than the ones between water molecules themselves. In this work, we studied the capability of sucrose moiety, attached to alkyl chains of different length, as a surface blocking agent at the water-interface and we compared its properties with those of polyethylenglycol, a well-known agent used for this purposes. Published studies in this topic mainly refer to the micellization process and the stability of mixed surfactant systems using glycosides. We are interested in the effect induced by the presence of sucrose monoesters at the interface (direct and reverse micelles) and at the palisade (mixtures with Triton X-100). We believe that the different functional group (ester), the position of alkyl chain (6-O) and the huge capability of sucrose to interact with water will dramatically change the water structuration at the interface and at the palisade, generating new possibilities for technological applications of these systems. Results Our time resolved and steady state fluorescence experiments in pure SEs micelles show that sucrose moieties are able to interact with a high number of water molecules promoting water structuration and increased viscosity. These results also indicate that the barrier formed by sucrose moieties on the surface of pure micelles is more effective than the polyoxyethylene palisade of Triton X-100

  7. Structuration in the interface of direct and reversed micelles of sucrose esters, studied by fluorescent techniques.

    Directory of Open Access Journals (Sweden)

    Catalina Sandoval

    Full Text Available Reactors found in nature can be described as micro-heterogeneous systems, where media involved in each micro-environment can behave in a markedly different way compared with the properties of the bulk solution. The presence of water molecules in micro-organized assemblies is of paramount importance for many chemical processes, ranging from biology to environmental science. Self-organized molecular assembled systems are frequently used to study dynamics of water molecules because are the simplest models mimicking biological membranes. The hydrogen bonds between sucrose and water molecules are described to be stronger (or more extensive than the ones between water molecules themselves. In this work, we studied the capability of sucrose moiety, attached to alkyl chains of different length, as a surface blocking agent at the water-interface and we compared its properties with those of polyethylenglycol, a well-known agent used for this purposes. Published studies in this topic mainly refer to the micellization process and the stability of mixed surfactant systems using glycosides. We are interested in the effect induced by the presence of sucrose monoesters at the interface (direct and reverse micelles and at the palisade (mixtures with Triton X-100. We believe that the different functional group (ester, the position of alkyl chain (6-O and the huge capability of sucrose to interact with water will dramatically change the water structuration at the interface and at the palisade, generating new possibilities for technological applications of these systems.Our time resolved and steady state fluorescence experiments in pure SEs micelles show that sucrose moieties are able to interact with a high number of water molecules promoting water structuration and increased viscosity. These results also indicate that the barrier formed by sucrose moieties on the surface of pure micelles is more effective than the polyoxyethylene palisade of Triton X-100. The

  8. Ultraviolet-visible light (UV-Vis)-reversible but fluorescence-irreversible chemosensor for copper in water and its application in living cells.

    Science.gov (United States)

    Huo, Fang-Jun; Yin, Cai-Xia; Yang, Yu-Tao; Su, Jing; Chao, Jian-Bin; Liu, Dian-Sheng

    2012-03-06

    An ultraviolet-visible light (UV-Vis)-reversible but fluorescence-irreversible chemosensor was developed for the detection of copper. Coordination between the probe, 2-pyridylaldehyde fluorescein hydrazone (FHP), and Cu(2+) gave a reversible UV-Vis response, Storage of the probe-Cu complex resulted in hydrolytic cleavage of the N═C bond, which released the fluorophore (ring-opened fluorescein hydrazine) and gave irreversible fluorescence. Thus, FHP becomes a multifunctional chemosensor, and its reversibility can be controlled by the reaction time. Cu(2+) in living cells could be detected using FHP and general fluorescence methods.

  9. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching.

    Directory of Open Access Journals (Sweden)

    Michele L Markwardt

    Full Text Available Cyan fluorescent proteins (CFPs, such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

  10. Synthesis of highly fluorescent silica nanoparticles in a reverse microemulsion through double-layered doping of organic fluorophores

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Hyojong, E-mail: hyojong@hallym.ac.kr; Pak, Joonsung [Hallym University, Department of Chemistry (Korea, Republic of)

    2013-05-15

    Water-soluble, highly fluorescent double-layered silica nanoparticles (FL-DLSN) have been successfully synthesized through a reverse (water-in-oil) microemulsion method. The microemulsion was prepared by mixing a surfactant (Brij35), co-surfactant, organic solvent, water, and fluorescein as an organic fluorophore. The sizes of the silica nanoparticles were successfully controlled in the reverse microemulsion using Brij35 by changing the water-to-Brij35 ratio and by adding HCl. Initially, tetraethylorthosilicate was hydrolyzed by adding NH{sub 4}OH as a catalyst and then polymerized to generate core fluorescent silica nanoparticles with fluorescein. 3-(Aminopropyl)triethoxysilane (APTS) was sequentially added into the reaction mixture, and reacted on the surface of pre-generated core silica nanoparticles to form the second layer in the form of a shell. The second silica layer that was derived from the condensation of APTS effectively protected the fluorescein dye within the silica matrix. This is a novel and simple synthetic approach to generate highly fluorescent, monodispersed silica nanoparticles by doping organic molecules into a silica matrix.Graphical Abstract.

  11. Kinetics of a single cross-bridge in familial hypertrophic cardiomyopathy heart muscle measured by reverse Kretschmann fluorescence

    Science.gov (United States)

    Mettikolla, Prasad; Calander, Nils; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Borejdo, Julian

    2010-01-01

    Familial hypertrophic cardiomyopathy (FHC) is a serious heart disease that often leads to a sudden cardiac death of young athletes. It is believed that the alteration of the kinetics of interaction between actin and myosin causes FHC by making the heart to pump blood inefficiently. We set out to check this hypothesis ex vivo. During contraction of heart muscle, a myosin cross-bridge imparts periodic force impulses to actin. The impulses are analyzed by fluorescence correlation spectroscopy (FCS) of fluorescently labeled actin. To minimize observation volume and background fluorescence, we carry out FCS measurements in surface plasmon coupled emission mode in a reverse Kretschmann configuration. Fluorescence is a result of near-field coupling of fluorophores excited in the vicinity of the metal-coated surface of a coverslip with the surface plasmons propagating in the metal. Surface plasmons decouple on opposite sides of the metal film and emit in a directional manner as far-field p-polarized radiation. We show that the rate of changes of orientation is significantly faster in contracting cardiac myofibrils of transgenic mice than wild type. These results are consistent with the fact that mutated heart muscle myosin translates actin faster in in vitro motility assays.

  12. Preparation of fluorescent organometallic porphyrin complex nanogels of controlled molecular structure via reverse-emulsion click chemistry.

    Science.gov (United States)

    Fu, Guo-Dong; Jiang, Hua; Yao, Fang; Xu, Li-Qun; Ling, Jun; Kang, En-Tang

    2012-09-26

    Here, we are the first to report a novel approach to preparing well-defined poly(ethylene glycol) (PEG) fluorescent nanogels, with well-defined molecular structures and desired functionalities via reverse (mini)emulsion copper(I)-catalyzed azide-alkyne cycloaddition (REM-CuAAC). Nanogels with hydroxyl groups and Ga-porphyrin complex (Ga-porphyrin-OH nanogels), as well as with Ga-porphyrin complex and folate functional groups (Ga-porphyrin-FA), are successfully prepared. Nanogels of 30 and 120 nm in diameter are obtained and they exhibit an emission maxima within the wavelength range 700-800 nm. The nanogels could find uses in near infrared (NIR) imaging attributable to their fluorescence and their functionality for cell affinity.

  13. High contrast imaging of reversibly switchable fluorescent proteins via temporally unmixed Multispectral Optoacoustic Tomography (tuMSOT)

    CERN Document Server

    Stiel, Andre C; Jiang, Yuanyuan; Ntziachristos, Vasilis; Razansky, Daniel; Westmeyer, Gil G

    2014-01-01

    Photocontrol of reversibly switchable fluorescent proteins (RSFPs) was used to program optoacoustic signal time courses that were temporally unmixed to increase the proteins contrast-to-noise-ratios (CNRs) in optoacoustic imaging. In this way, two variants of the RSFP Dronpa with very similar optoacoustic spectra could be readily discriminated in the presence of highly absorbing blood. Addition of temporal unmixing to multispectral optoacoustic tomography (tuMSOT) in conjunction with synthetic or genetically controlled photochromic contrast agents and customized photoswitching schedules can increase the performance of multiplexed and high contrast molecular optoacoustic imaging.

  14. A reversible multi-stimuli-responsive fluorescence probe and the design for combinational logic gate operations.

    Science.gov (United States)

    Xia, Hongyan; Xu, Yangyang; Yang, Guang; Jiang, Hao; Zou, Gang; Zhang, Qijin

    2014-02-01

    Here, a novel multi-stimuli-responsive fluorescence probe is developed by incorporating spiropyran group into the coumarin-substituted polydiacetylene (PDA) vesicles. The fluorescence of PDA can be turned on upon heating, and can be quenched upon exposure to UV light irradiation or pH stimuli owing to the fluorescene resonance energy transfer (FRET) between the red-phase PDA and the open merocyanine (MC) form of spiropyran. Moreover, we have designed and experimentally realized a set of logic gate operations for the first time based on the fluorescence modulation of the designed system upon thermal, photo, and pH stimuli. This novel type of resettable logic gates augur well for practical applications in information storage, optical recording, and sensing in complicated microenvironments. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

    Science.gov (United States)

    Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

  16. Determination of Fluorescent Whitening Agents in Paper Materials by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jeong Soo; Kim, Keon [Korea Univ., Seoul (Korea, Republic of); Kim, Do Hwan [Daegu Univ., Gyeongsan (Korea, Republic of)

    2012-12-15

    A simple method was developed for the analysis of seven stilbene-type fluorescent whitening agents (FWAs) in paper materials by ion-pair reversed-phase high-performance liquid chromatography with fluorescence detection. These stilbene-type FWAs included two disulfonate, two tetrasulfonate, and three hexasulfonate compounds. After optimization of chromatographic conditions, the FWAs were satisfactorily separated using a reversed-phase column (RP-18) with the following isocratic mobile phase: methanol-water (60:40) containing 17.5 mM TBABr and 10 mM citrate buffer (pH = 7.0). The calibration plot was linear in the range from 5 to 500 ng/mL for two disulfo-FWAs and from 1 to 500 ng/mL for the other five FWAs. Precision levels of the calibration curve as indicated by RSD of response factors were 1.2 and 8.1%. Limits of quantitation (LOQ) ranged from 1.2 to 11 ng/mL.

  17. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-10-15

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.

  18. Development of Conventional and Real-Time Reverse Transcription Polymerase Chain Reaction Assays to Detect Tembusu Virus in Culex tarsalis Mosquitoes

    Science.gov (United States)

    2014-08-11

    appropri- ate preventive measures can be taken to prevent morbidity and mortality associated with infection with these pathogens. Development of...field-based real-time quantitative RT-PCR (qRT-PCR) assays for rapid detection of virus-infected mos- quitoes would be a great assistance to preventive ...specimens were propagated in primary duck embryo, C6/36 ( Aedes albopictus), or Vero (African green monkey kidney) cells to produce stock viruses for

  19. 3种重要虫媒病毒的RT-PCR检测%Detection of three important arbovirus strains by reverse transcription polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    于曼; 秦鄂德; 邓永强; 赵月峨; 范宝昌; 彭文明; 杨保安; 司炳银; 祝庆余

    2003-01-01

    目的:建立快速、敏感、特异的虫媒病毒RT-PCR检测方法.方法:采用改进的核酸制备方法,分别从感染的乳鼠脑和细胞培养上清液中提取病毒RNA,再用RT-PCR和套式PCR方法进行检测.结果:通过对RT-PCR反应条件的优化,用3种虫媒病毒的特异引物均可从不同稀释度的感染小鼠脑和细胞上清中提取的病毒RNA扩增出特异的目的基因片段.甲病毒属的东部马脑炎病毒和西部马脑炎病毒之间,及与黄病毒属的黄热病毒均无交叉反应,表明建立的RT-PCR检测方法特异性强.套式PCR与RT-PCR比较可提高检测敏感性10~10 000倍.结论:建立的RT-PCR和套式PCR法可用于3种虫媒病毒的快速检测.

  20. Dengue virus detection by using reverse transcription-polymerase chain reaction in saliva and progeny of experimentally infected Aedes albopictus from Brazil

    Directory of Open Access Journals (Sweden)

    Márcia Gonçalves de Castro

    2004-12-01

    Full Text Available Oral susceptibility and vertical transmission of dengue virus type 2 (DENV-2 in an Aedes albopictus sample from Rio de Janeiro was estimated. The infection (36.7% and transmission (83.3% rates for Ae. albopictus were higher than those of an Ae. aegypti colony used as control, 32.8 and 60%, respectively. Fourth instar larvae and females descendants of 48.5 and 39.1% of experimentally infected Ae. albopictus showed to harbor the virus. The oral susceptibility and the high capacity to assure vertical transmission exhibited by Ae. albopictus from Brazil reinforce that this species may play a role in the maintenance of the virus in nature and be a threat for dengue control in the country.

  1. A multiple reverse transcription-polymerase chain reaction assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees

    Science.gov (United States)

    Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, in...

  2. Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs

    DEFF Research Database (Denmark)

    Reid, S.M.; Paton, D.J.; Wilsden, G.;

    2004-01-01

    of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs....

  3. MONITORING MYCOTOXIN PRODUCTION AT THE GENETIC LEVEL ON VARIOUS GROWTH SUBSTRATES USING QUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION?EXPERIMENT DESIGN

    Science.gov (United States)

    The paper describes a method of analyzing the production of mycotoxins at the genetic level by monitoring the intracellular levels of messenger RNA (mRNA). Initial work will focus on threshing out the mycotoxin gene clusters in Stachybotrys chartarum followed by analysis of toxin...

  4. Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile

    Directory of Open Access Journals (Sweden)

    David Tapia

    2017-07-01

    Conclusions: Overall, the ring trial showed high values of sensitivity and specificity, with some problems of repeatability and inter-laboratory variability. This last issue needs to be addressed in order to allow harmonized diagnostic of IPNV within the country. We recommend the use of the NRL methods as validated and reliable qRT-PCR protocols for the detection of IPNV.

  5. Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs

    DEFF Research Database (Denmark)

    Reid, S.M.; Paton, D.J.; Wilsden, G.

    2004-01-01

    Automated real-time RT-PCR was evaluated as a diagnostic tool for swine vesicular disease virus (SVDV) infection on a range of samples (vesicular epithelium, serum, nasal swabs, faeces) from four inoculated and three in-contact pigs over a period of 28 days. Traditional diagnostic procedures (virus....... The RT-PCR and virus isolation were generally comparable in detecting SVDV in the serum and nasal swabs from inoculated and in-contact pigs up to day 6 after infection; it was possible, however, to isolate virus for a longer period from the faeces of a few pigs. This suggested that further optimization...... of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs....

  6. 介绍一种快速反转录聚合酶链反应%Introduction of an access of reverse transcription polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    张敬如

    2001-01-01

    目的介绍一种特异、高效的快速PCR方法。方法总RNA分成2份,采用相同反应体系,分别用普通PCR方法和快速PCR方法同时扩增FasL。结果虽然两种方法均能成功扩增FasL基因,但普通PCR方法耗时3h48min,而且还存在非特异性片段;而快速PCR方法耗时仅2h6min,而且没有出现非特异片段。结论快速PCR方法不仅极大的缩短了反应时间,同时提高了反应的特异性。

  7. MONITORING MYCOTOXIN PRODUCTION AT THE GENETIC LEVEL ON VARIOUS GROWTH SUBSTRATES USING QUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION?EXPERIMENT DESIGN

    Science.gov (United States)

    The paper describes a method of analyzing the production of mycotoxins at the genetic level by monitoring the intracellular levels of messenger RNA (mRNA). Initial work will focus on threshing out the mycotoxin gene clusters in Stachybotrys chartarum followed by analysis of toxin...

  8. Rapid detection and quantification of Ebola Zaire virus by one-step real-time quantitative reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Ro, Young-Tae; Ticer, Anysha; Carrion, Ricardo; Patterson, Jean L

    2017-04-01

    Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 10(2) to 10(3) copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  9. Reverse engineering the ancient ceramic technology based on X-ray fluorescence spectromicroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Sciau, Philippe; Leon, Yoanna; Goudeau, Philippe; Fakra, Sirine C.; Webb, Sam; Mehta, Apurva

    2011-07-06

    We present results of X-ray fluorescence (XRF) microprobe analyses of ancient ceramic cross-sections aiming at deciphering the different firing protocols used for their production. Micro-focused XRF elemental mapping, Fe chemical mapping and Fe K-edge X-ray absorption near edge structure spectroscopy were performed on pre-sigillata ceramics from southern Gaul, and terra Sigillata vessels from Italy and southern Gaul. Pieces from the different workshops and regions showed significant difference in the starting clay material, clay conditioning and kiln firing condition. By contrast, sherds from the same workshop exhibited more subtle differences and possible misfirings. Understanding the precise firing conditions and protocols would allow recreation of kilns for various productions. Furthermore, evolution and modification of kiln design would shed some light on how ancient potters devised solutions to diverse technological problems they encountered.

  10. Thiamin analysis in red wine by fluorescence reverse phase-HPLC.

    Science.gov (United States)

    Liddicoat, Callum; Hucker, Barry; Liang, Hao; Vriesekoop, Frank

    2015-06-15

    The derivatization of thiamin vitamers to their respective thiochrome by ferricyanide to facilitate fluorescence detection following separation by HPLC provides a powerful analytical tool. However the polyphenolic compounds in red wine readily interact with ferricyanide, reducing the effectiveness of ferricyanide oxidation in the derivatization of thiamin. We describe a method to facilitate the removal of polyphenolic compounds that interfere with the ferricyanide derivatization of thiamin. Polyvinylpolypyrrolidone afforded the total removal of phenolic compounds from red wines and allowed a spike recovery of thiamin vitamers (101% for thiamin; 104% for TMP; and 100% for TDP) in a wide range of red wines. This research found that Merlot styles of red wine contained the highest concentration of total thiamin (29.01 ng/mL) while Pinot Noir wines contained the lowest total concentration (8.27 ng/mL). Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics

    Science.gov (United States)

    Umezawa, Keitaro; Yoshida, Masafumi; Kamiya, Mako; Yamasoba, Tatsuya; Urano, Yasuteru

    2017-03-01

    Alterations in glutathione (GSH) homeostasis are associated with a variety of diseases and cellular functions, and therefore, real-time live-cell imaging and quantification of GSH dynamics are important for understanding pathophysiological processes. However, existing fluorescent probes are unsuitable for these purposes due to their irreversible fluorogenic mechanisms or slow reaction rates. In this work, we have successfully overcome these problems by establishing a design strategy inspired by Mayr's work on nucleophilic reaction kinetics. The synthesized probes exhibit concentration-dependent, reversible and rapid absorption/fluorescence changes (t1/2 = 620 ms at [GSH] = 1 mM), as well as appropriate Kd values (1-10 mM: within the range of intracellular GSH concentrations). We also developed FRET-based ratiometric probes, and demonstrated that they are useful for quantifying GSH concentration in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds.

  12. Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm

    Science.gov (United States)

    El Khatib, Mariam; Martins, Alexandre; Bourgeois, Dominique; Colletier, Jacques-Philippe; Adam, Virgile

    2016-01-01

    Phototransformable fluorescent proteins are central to several nanoscopy approaches. As yet however, there is no available variant allowing super-resolution imaging in cell compartments that maintain oxidative conditions. Here, we report the rational design of two reversibly switchable fluorescent proteins able to fold and photoswitch in the bacterial periplasm, rsFolder and rsFolder2. rsFolder was designed by hybridisation of Superfolder-GFP with rsEGFP2, and inherited the fast folding properties of the former together with the rapid switching of the latter, but at the cost of a reduced switching contrast. Structural characterisation of the switching mechanisms of rsFolder and rsEGFP2 revealed different scenarios for chromophore cis-trans isomerisation and allowed designing rsFolder2, a variant of rsFolder that exhibits improved switching contrast and is amenable to RESOLFT nanoscopy. The rsFolders can be efficiently expressed in the E. coli periplasm, opening the door to the nanoscale investigation of proteins localised in hitherto non-observable cellular compartments. PMID:26732634

  13. A Structural Basis for Reversible Photoswitching of Absorbance Spectra in Red Fluorescent Protein rsTagRFP

    Energy Technology Data Exchange (ETDEWEB)

    Pletnev, Sergei; Subach, Fedor V.; Dauter, Zbigniew; Wlodawer, Alexander; Verkhusha, Vladislav V. (Einstein); (NCI)

    2012-09-05

    rsTagRFP is the first monomeric red fluorescent protein (FP) with reversibly photoswitchable absorbance spectra. The switching is realized by irradiation of rsTagRFP with blue (440 nm) and yellow (567 nm) light, turning the protein fluorescence ON and OFF, respectively. It is perhaps the most useful probe in this color class that has yet been reported. Because of the photoswitchable absorbance, rsTagRFP can be used as an acceptor in photochromic Foerster resonance energy transfer. Yellow FPs, YPet and mVenus, are demonstrated to be excellent photochromic Foerster resonance energy transfer donors for the rsTagRFP acceptor in its fusion constructs. Analysis of X-ray structures has shown that photoswitching of rsTagRFP is accompanied by cis-trans isomerization and protonation/deprotonation of the chromophore, with the deprotonated cis- and protonated trans-isomers corresponding to its ON and OFF states, respectively. Unlike in other photoswitchable FPs, both conformers of rsTagRFP chromophore are essentially coplanar. Two other peculiarities of the rsTagRFP chromophore are an essentially hydrophobic environment of its p-hydroxyphenyl site and the absence of direct hydrogen bonding between this moiety and the protein scaffold. The influence of the immediate environment on rsTagRFP chromophore was probed by site-directed mutagenesis. Residues Glu145 and His197 were found to participate in protonation/deprotonation of the chromophore accompanying the photoswitching of rsTagRFP fluorescence, whereas residues Met160 and Leu174 were shown to spatially restrict chromophore isomerization, favoring its radiative decay.

  14. Dual-stimuli responsive and reversibly activatable theranostic nanoprobe for precision tumor-targeting and fluorescence-guided photothermal therapy

    Science.gov (United States)

    Zhao, Xu; Yang, Cheng-Xiong; Chen, Li-Gong; Yan, Xiu-Ping

    2017-05-01

    The integrated functions of diagnostics and therapeutics make theranostics great potential for personalized medicine. Stimulus-responsive therapy allows spatial control of therapeutic effect only in the site of interest, and offers promising opportunities for imaging-guided precision therapy. However, the imaging strategies in previous stimulus-responsive therapies are `always on' or irreversible `turn on' modality, resulting in poor signal-to-noise ratios or even `false positive' results. Here we show the design of dual-stimuli-responsive and reversibly activatable nanoprobe for precision tumour-targeting and fluorescence-guided photothermal therapy. We fabricate the nanoprobe from asymmetric cyanine and glycosyl-functionalized gold nanorods (AuNRs) with matrix metalloproteinases (MMPs)-specific peptide as a linker to achieve MMPs/pH synergistic and pH reversible activation. The unique activation and glycosyl targetibility makes the nanoprobe bright only in tumour sites with negligible background, while AuNRs and asymmetric cyanine give synergistic photothermal effect. This work paves the way to designing efficient nanoprobes for precision theranostics.

  15. Validation of a multiplex reverse transcriptase PCR ELISA for the detection of 19 respiratory tract pathogens

    NARCIS (Netherlands)

    Puppe, W.; Weigl, J.; Groendahl, B.; Knuf, M.; Rockahr, S.; von Bismarck, P.; Aron, G.; Niesters, H. G. M.; Osterhaus, A. D. M. E.; Schmitt, H. -J.

    2013-01-01

    Introduction Since acute respiratory tract infections inflict a high burden of disease in children worldwide, a multiplex reverse transcription polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR ELISA) to detect 19 different respiratory pathogens was developed and vali

  16. Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators.

    Science.gov (United States)

    Kotera, Ippei; Iwasaki, Takuya; Imamura, Hiromi; Noji, Hiroyuki; Nagai, Takeharu

    2010-02-19

    Fluorescent protein (FP)-based Forster resonance energy transfer (FRET) technology is useful for development of functional indicators to visualize second messenger molecules and activation of signaling components in living cells. However, the design and construction of the functional indicators require careful optimization of their structure at the atomic level. Therefore, routine procedures for constructing FRET-based indicators currently include the adjustment of the linker length between the FPs and the sensor domain and relative dipole orientation of the FP chromophore. Here we report that, in addition to these techniques, optimization of the dimerization interface of Aequorea FPs is essential to achieve the highest possible dynamic range of signal change by FRET-based indicators. We performed spectroscopic analyses of various indicators (cameleon, TN-XL, and ATeam) and their variants. We chose variants containing mutant FPs with different dimerization properties, i.e., no, weak, or enhanced dimerization of the donor or acceptor FP. Our findings revealed that the FPs that dimerized weakly yielded high-performance FRET-based indicators with the greatest dynamic range.

  17. Quantification of mRNA Levels by Fluorescently Labelled Reverse Transcription Competitive PCR

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A reproducible,quantitative,non-radioactive method for the analysis of mRNA expression is described.After RNA preparation and cDNA synthesi s,the cDNA was co-amplified with an internal standard in the same PCR system.Th e PCR products containing both targen and internal standard amplificates were el ectrophoresed and detected on an ABI 377 DNA Sequencer.For each sample,β-actin was also quantified by an identical procedure to compensate for relative differ ences between samples in the integrity of the individual RNA samples and for var iations in reverse transcription.Due to the linear relationship between cDNA con tent and PCR product ratio of target cDNA template and competitive standard,a si ngle PCR reaction was sufficient for quantification of a sample.The experimental results showed that the method is a mRNA quantitative RT-PCR method with high sensitivity and good reproducibility.It can be used in large-scale accurate qu antitative analyses of mRNA expression of any gene.

  18. Reversibly monitoring oxidation and reduction events in living biological systems: Recent development of redox-responsive reversible NIR biosensors and their applications in in vitro/in vivo fluorescence imaging.

    Science.gov (United States)

    Chu, Tian-Shu; Lü, Rui; Liu, Bai-Tong

    2016-12-15

    Reactive oxygen species (ROS) and changes in their redox cycles have great therapeutic potential for treating serious redox-related human diseases such as acute and chronic inflammation, diabetes, cancer and neurodegenerative disorders. This article presents a survey of the recently (2011-2016) developed NIR small-molecule biosensors for reversibly monitoring oxidation and reduction events in living cells and small animals through in vitro/in vivo fluorescence imaging. Emission and absorption profile, design strategy and fluorescence sensing mechanism, ROS selectivity and sensitivity, reversibility, ability of subcellular location and cytotoxicity are discussed for the NIR small-molecule biosensors capable of quantitatively, continuously and reversibly detecting transient ROS burst and redox changes at cellular level.

  19. Detection of norovirus, sapovirus, and human astrovirus in fecal specimens using a multiplex reverse transcription-PCR with fluorescent dye-labeled primers.

    Science.gov (United States)

    Shigemoto, Naoki; Fukuda, Shinji; Tanizawa, Yukie; Kuwayama, Masaru; Ohara, Sachiko; Seno, Masato

    2011-05-01

    We applied a multiplex reverse transcription-PCR with fluorescent dye-labeled primers (fluorescent multiplex RT-PCR) for noroviruses (NoV), sapovirus (SaV), and human astrovirus (HAstV) to diagnose 71 outbreaks of acute gastroenteritis during July 2007 and May 2010 in Hiroshima prefecture. In this assay, the green, red, yellow, and blue fluorescence for NoV genogroup I, NoV genogroup II, SaV, and HAstV, respectively, were indicated on an agarose gel under ultraviolet light. In 61 virus-positive outbreaks confirmed by fluorescent multiplex RT-PCR, detection rates of outbreaks for NoVs, SaV, and HAstV were 96.7%, 3.3%, and 0%, respectively. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

  20. Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization.

    Science.gov (United States)

    Reinen, Jelle; Kool, Jeroen; Vermeulen, Nico P E

    2008-04-01

    We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor alpha (ERalpha) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17beta-estradiol, 17alpha-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERalpha. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERalpha. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence.

  1. Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells.

    Science.gov (United States)

    Chen, Jianwei; Jiang, Xiqian; Carroll, Shaina L; Huang, Jia; Wang, Jin

    2015-12-18

    Density functional theory (DFT) was applied to study the thermodynamics and kinetics of reversible thiol-Michael addition reactions. M06-2X/6-31G(d) with the SMD solvation model can reliably predict the Gibbs free energy changes (ΔG) of thiol-Michael addition reactions with an error of less than 1 kcal·mol(-1) compared with the experimental benchmarks. Taking advantage of this computational model, the first reversible reaction-based fluorescent probe was developed that can monitor the changes in glutathione levels in single living cells.

  2. Synthesis and photochromism of naphthopyrans bearing naphthalimide chromophore: predominant thermal reversibility in color-fading and fluorescence switch.

    Science.gov (United States)

    Song, Liwen; Yang, Yuheng; Zhang, Qiong; Tian, He; Zhu, Weihong

    2011-12-15

    Two novel photochromic naphthopyrans containing naphthalimide moieties (Nip1 and Nip2) were studied in solution under flash photolysis conditions, exhibiting highly photochromic response, rapid thermal bleaching rate and good fatigue-resistance. Owing to the different N-substituted imide groups at the naphthalimide units, the thermal bleaching rate of Nip2 bearing phenyl on the naphthalimide unit is found to be approximately 2 times that of Nip1 bearing n-butyl, indicating that the photochromic properties can be modulated with introduction of different functional groups on the naphthalimide unit. In Nip1 and Nip2, the strong electron-withdrawing effect of the imide group incorporated at the naphthalimide moiety maintains several merits: (i) shifting absorption bands to longer wavelength, (ii) beneficial to an enhancement in the ratio of transoid-cis (TC) isomer and an increase in the transformation rate from transoid-trans (TT) to TC with respect to reference compound NP, and (iii) resulting in a preferable color bleaching rate and fading absolutely to their colorless state with thermal reversibility. As demonstrated in the system of NP, the slow transformation process from TT to TC might be the predominant dynamic step in thermal back process, leading to the residual color of NP being only faded to its original colorless state by visible light irradiation. The optical densities of colored forms for Nip1 and Nip2 are dependent upon the intensity of incident light, ensuring a possible application in the manufacture of ophthalmic lenses and smart windows. Moreover, the fluorescence of Nip1 and Nip2 can be switched on and off by photoinduced conversion between the closed and open forms.

  3. Cellular Activation of the Self-Quenched Fluorescent Reporter Probe in Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Alexei A. Bogdanov, Jr.

    2002-01-01

    Full Text Available The effect of intralysosomal proteolysis of near-infrared fluorescent (NIRF self-quenched macromolecular probe (PGC-Cy5.5 has been previously reported and used for tumor imaging. Here we demonstrate that proteolysis can be detected noninvasively in vivo at the cellular level. A codetection of GFP fluorescence (using two-photon excitation and NIRF was performed in tumor-bearing animals injected with PGC-Cy5.5. In vivo microscopy of tumor cells in subdermal tissue layers (up to 160 μm showed a strong Cy5.5 dequenching effect in GFP-negative cells. This observation was corroborated by flow cytometry, sorting, and reverse transcription polymerase chain reaction analysis of tumor-isolated cells. Both GFP-positive (81% total and GFP-negative (19% total populations contained Cy5.5-positive cells. The GFP-negative cells were confirmed to be host mouse cells by the absence of rat cathepsin mRNA signal. The subfraction of GFPnegative cells (2.5-3.0% had seven times higher NIRF intensity than the majority of GFP-positive or GFPnegative cells (372 and 55 AU, respectively. Highly NIRF-positive, FP-negative cells were CD45-and MAC3-positive. Our results indicate that: 1 intracellular proteolysis can be imaged in vivo at the cellular level using cathepsin-sensitive probes; 2 tumor-recruited cells of hematopoetic origin participate most actively in uptake and degradation of long-circulating macromolecular probes.

  4. Sry-negative XX sex reversal in a French Bulldog.

    Science.gov (United States)

    Campos, M; Moreno-Manzano, V; García-Roselló, M; García-Roselló, E

    2011-02-01

    Here, we describe a 3-month-old XX male French Bulldog. The diagnosis was based on the clinical signs, gonadal histology and cytogenetic analysis. Additionally, the dog was confirmed to be Sry negative by semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR). Canine Sry-negative XX sex reversal is a disorder of gonadal development where individuals who have a female karyotype develop testes or ovotestes. To our knowledge, this case is the first XX male sex reversion described in a French Bulldog.

  5. A bacterial community analysis using reverse transcription (RT) PCR which detects the bacteria with high activity in a wastewater treatment reactor

    Science.gov (United States)

    This research used reverse transcription polymerase chain reaction (RT-PCR) method to help detect active bacteria in a single-tank deammonification reactor combining partial nitritation and anammox. The single-tank aerobic deammonification reactor effectively removed the ammonia in anaerobically di...

  6. One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus.

    Science.gov (United States)

    Li, Jin-yu; Wei, Qi-wei; Liu, Yong; Tan, Xin-qiu; Zhang, Wen-na; Wu, Jian-yan; Charimbu, Miriam Karwitha; Hu, Bai-shi; Cheng, Zhao-bang; Yu, Cui; Tao, Xiao-rong

    2013-11-01

    Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV.

  7. Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification

    Directory of Open Access Journals (Sweden)

    Joojin Jeong

    2015-09-01

    Full Text Available The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

  8. Analysis of electrophoretic soil humic acids fractions by reversed-phase high performance liquid chromatography with on-line absorbance and fluorescence detection.

    Science.gov (United States)

    Trubetskoj, Oleg A; Richard, Claire; Guyot, Ghislain; Voyard, Guillaume; Trubetskaya, Olga E

    2012-06-22

    A combination of reversed-phase high performance liquid chromatography (RP HPLC) with on-line absorbance and fluorescence detection was used for analysis of chernozem soil humic acids (HAs) and their fractions A, B and C+D with different electrophoretic mobility (EM) and molecular size (MS). Samples were injected onto the column at the identical volume and absorbance. All chromatograms exhibit the resolution of seven peaks. The estimation of relative recovery of HAs and fractions from the reverse-phase column has been done. High MS fraction A, which possesses the low EM, is essentially more hydrophobic (73% of the fraction amount remained adsorbed on the column) and aliphatic than medium MS and EM fraction B (33% of the fraction amount remained adsorbed on the column). The most hydrophilic and aromatic properties belong to low MS fraction C+D, which possess the highest EM and practically was not adsorbed on the column. The hydrophobicity of the bulk HAs lies within the range of fractions hydrophobicity. The absorption spectra of bulk HAs, electrophoretic fractions A, B, C+D and corresponding RP HPLC peaks were featureless but had differences in the values of absorbance ratio at 300 and 400 nm (A3/A4). For fractions A and B this ratio gradually decreased from peak 1 to 7 (from 3.05 to 2.80 and 3.00 to 2.40, respectively). This trend was less pronounced in HAs and practically absent in fraction C+D, where ratio A3/A4 varied within a small range. The strong relationship between fluorescence properties, EM, MS, polarity and aliphaticity/aromaticity of HAs fractions was found. Humic and protein-like fluorescence had different polarity nature. The protein-like fluorescence is located in humic material which irreversibly adsorbed on the reverse-phase column and not subjected to RP HPLC characterization. The humic-like fluorescence at Ex/Em 270/450 nm is mostly located in the hydrophilic peak of low MS fraction C+D. Taking into account that high MS fraction A consisted

  9. Ratiometric fluorescence sensing of sugars via a reversible disassembly and assembly of the peptide aggregates mediated by sugars.

    Science.gov (United States)

    Neupane, Lok Nath; Han, Song Yee; Lee, Keun-Hyeung

    2014-06-01

    An amphiphilic dipeptide (1) bearing pyrene and phenylboronic acid was demonstrated as a unique example of a ratiometric sensing system for sugars by reversibly converting the peptide aggregates into the monomer form of the complex with sugars in aqueous solutions.

  10. Impact of organic fractions identified by SEC and fluorescence EEM on the hydraulic reversibility of ultrafiltration membrane fouling by secondary effluents

    KAUST Repository

    Haberkampa, Jens

    2011-05-01

    Loss of membrane filtration performance due to organic fouling is still a significant drawback for the application of low-pressure membranes in tertiary wastewater treatment. The present study investigates the relevance of different organic fractions present in secondary effluents in terms of hydraulically reversible and irreversible fouling of hollow-fibre ultrafiltration membranes. A good correlation between the hydraulically reversible filtration resistance and the total organic biopolymer concentration according to size exclusion chromatography (SEC) was observed. Qualitatively biopolymers consist mainly of polysaccharides as well as proteins with high molecular weight. Polysaccharides are retained by the membrane pores, but can be removed by simple UF backwashing. On the other hand, fluorescence excitation-emission matrix (EEM) analysis indicates that the extent of the hydraulically irreversible fouling correlates with the presence of protein-like substances. Removal of protein-like substances by biological slow sand filtration or chemical coagulation results in the significant reduction of the hydraulically irreversible fouling, which is presumably due to proteins in the molecular range of biopolymers. In contrast to the comparatively low sensitivity of colorimetric methods for the analysis of proteins and polysaccharides, the combined application of size exclusion chromatography and fluorescence EEM analysis is a promising tool for the determination of the organic fouling propensity of secondary effluents. ©2011 Desalination Publications. All rights reserved.

  11. Real-time determination of human telomerase reverse transcriptase mRNA in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Li-Hua Hu; Feng-Hua Chen; Yi-Rong Li; Lin Wang

    2004-01-01

    AIM: To set up a real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay,to detect human telomerase reverse transcriptase (hTERT)messenger RNA in gastric carcinomas, and to evaluate quantitative determination of hTERT mRNA in the diagnostic value of gastric carcinomas, and to analyze the correlation between the expression level of hTERT mRNA and dinicopathological parameters in patients with gastric cancer.METHODS: A real-time quantitative RT-PCR (RQ-PCR)based on TaqMan fluorescence methodoloogy and the LightCyder system was used to quantify the full range of hTERT mRNA copy numbers in 35 samples of gastric carcinomas and corresponding adjacent non-cancerous tissues. The normalized hTERT (NhTERT) was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100× (hTERT/GAPDH) ratio. Variables were analyzed by the Student's t-test, χ2 test and Fisher's exact test.RESULTS: NhTERT from gastric carcinomas and corresponding adjacent non-cancerous tissues was 6.27±0.89 and 0.93±0.18,respectively (t= 12.76, P<0.001). There was no significant association between gastric cancer hTERT mRNA expression level and patient's age, gender, tumor size, location and stage (pTNM), but a significant correlation was found between hTERT mRNA expression level in gastric carcinomas and the degree of differentiation.CONCLUSION: Quantitative determination of hTERT mRNA by RQ-PCR is a rapid and sensitive method. hTERT might be a potential biomarker for the early detection of gastric cancer.

  12. X-ray Free Electron Laser Determination of Crystal Structures of Dark and Light States of a Reversibly Photoswitching Fluorescent Protein at Room Temperature

    Directory of Open Access Journals (Sweden)

    Christopher D. M. Hutchison

    2017-09-01

    Full Text Available The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on state at 1.9 Angstrom resolution and the trans (off state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form.

  13. A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Kyung oh [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Biomedical Sciences, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Tumor Microenvironment Global Core Research Center, Seoul National University (Korea, Republic of); Youn, Hyewon, E-mail: hwyoun@snu.ac.kr [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Tumor Microenvironment Global Core Research Center, Seoul National University (Korea, Republic of); Cancer Imaging Center, Seoul National University Hospital, Seoul (Korea, Republic of); Kim, Seung Hoo [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Kim, Young-Hwa [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Biomedical Sciences, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Kang, Keon Wook [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Chung, June-Key, E-mail: jkchung@snu.ac.kr [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Biomedical Sciences, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Tumor Microenvironment Global Core Research Center, Seoul National University (Korea, Republic of)

    2016-08-26

    Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and {sup 64}Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescence signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.

  14. Expression, purification, crystallization and preliminary X-ray analysis of eCGP123, an extremely stable monomeric green fluorescent protein with reversible photoswitching properties.

    Science.gov (United States)

    Don Paul, Craig; Traore, Daouda A K; Byres, Emma; Rossjohn, Jamie; Devenish, Rodney J; Kiss, Csaba; Bradbury, Andrew; Wilce, Matthew C J; Prescott, Mark

    2011-10-01

    Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74.63, b = 75.38, c = 84.51 Å, α = 90.96, β = 89.92, γ = 104.03°. The Matthews coefficient (V(M) = 2.26 Å(3) Da(-1)) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules.

  15. Synthesis of fluorescent label, DBD-beta-proline, and the resolution efficiency for chiral amines by reversed-phase chromatography.

    Science.gov (United States)

    Min, Jun Zhe; Toyo'oka, Toshimasa; Kato, Masaru; Fukushima, Takeshi

    2005-01-01

    DBD-d(and l)-beta-proline, new fluorescent chiral derivatization reagents, were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7- fl uoro-2,1,3-benzoxadiazole (DBD-F) with beta-proline. The racemic mixture synthesized was separated by a chiral stationary phase (CSP) column, Chiralpak AD-H, with n-hexane-EtOH-TFA-diethylamine (70:30:0.1:0.1) as the mobile phase. The dl-forms were decided according to the results obtained from a circular dichroism (CD) detector after separation by the CSP column. The fractionated enantiomers reacted with chiral amine to produce a couple of diastereomers. The labeling proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine as the activation reagents. The reaction conditions were mild and no racemization occurred during the diastereomer formation. The resulting diastereomers fluoresced at around 570 nm (excitation at around 460 nm). Good linearity of the calibration curves was obtained in the range 1-75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was compared with the diastereomers derived from DBD-d(or l)-proline. The resolution values (Rs) obtained from the diastereomers of three chiral amines with DBD-d(or l)-beta-proline were higher than those derived from DBD-d(or l)-proline, e.g. dl-phenylalanine methylester (dl-PAME), 2.23 vs 1.37; (R)(S)-1-phenylethylamine [(R)(S)-PEA], 2.09 vs 1.13; and (R)(S)-1-(1-naphthyl)ethylamines [(R)(S)-NEA], 5.19 vs 1.23. The results suggest that the position of COOH group on pyrrolidine moiety in the structures is one of the important factors for the efficient separation of a couple of the diastereomers.

  16. Evaluation of the Sofia Influenza A + B fluorescent immunoassay for the rapid diagnosis of influenza A and B.

    Science.gov (United States)

    Hazelton, Briony; Nedeljkovic, Gordana; Ratnamohan, V Mala; Dwyer, Dominic E; Kok, Jen

    2015-01-01

    Rapid influenza diagnostic tests (RIDTs) can facilitate the appropriate prescription of antivirals for influenza, obviate the need for unnecessary testing and antibacterial agents and allow the implementation of infection control measures. However, the reported sensitivities and specificities of different RIDTs vary widely in clinical settings, as does assay ability to distinguish between influenza types and subtypes. To evaluate the performance of the Sofia Influenza A + B fluorescent immunoassay (FIA) for the detection of influenza A and B during the 2013 Southern Hemisphere influenza season, a total of 209 consecutive respiratory tract swabs from adult patients with an influenza-like illness were tested by both Sofia Influenza A + B and an in-house real-time, reverse transcription-polymerase chain reaction (RT-PCR) assay. Compared to RT-PCR, the sensitivity and specificity of the Sofia Influenza A + B FIA for detection of influenza A was 72.4% and 98.3%, respectively. Too few influenza B positive samples were available during the study to accurately assess the Sofia's performance for influenza B detection. The sensitivity of Sofia Influenza A + B FIA for both influenza A and B detection correlated with the amount of influenza RNA present in the sample as indicated indirectly by the RT-PCR cycle threshold (Ct ). In conclusion, the Sofia Influenza A + B FIA continues to perform well as a RIDT with the circulating influenza strains of the 2013 Southern Hemisphere influenza season.

  17. Photoisomerization and proton transfer in the forward and reverse photoswitching of the fast-switching M159T mutant of the Dronpa fluorescent protein.

    Science.gov (United States)

    Kaucikas, Marius; Tros, Martijn; van Thor, Jasper J

    2015-02-12

    The fast-switching M159T mutant of the reversibly photoswitchable fluorescent protein Dronpa has an enhanced yield for the on-to-off reaction. The forward and reverse photoreactions proceed via cis-trans and trans-cis photoisomerization, yet protonation and deprotonation of the hydroxyphenyl oxygen of the chromophore is responsible for the majority of the resulting spectroscopic contrast. Ultrafast visible-pump, infrared-probe spectroscopy was used to detect the picosecond, nanosecond, as well as metastable millisecond intermediates. Additionally, static FTIR difference measurements of the Dronpa-M159T mutant correspond very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone chromophore in the cis anion and trans neutral forms in the bright "on" and dark "off" states, respectively. Green excitation of the on state is followed by dominant radiative decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1 ns, and additionally reveals spectral changes belonging to the species decaying with a 1.1 ns time constant, associated with both protein and chromophore modes. A 1 ms measurement of the on state identifies bleach features that correspond to those seen in the static off-minus-on Fourier transform infrared (FTIR) difference spectrum, indicating that thermal protonation of the hydroxyphenyl oxygen proceeds within this time window. Blue excitation of the off state directly resolves the formation of the primary photoproduct with 0.6 and 14 ps time constants, which is stable on the nanosecond time scale. Assignment of the primary photoproduct to the cis neutral chromophore in the electronic ground state is supported by the frequency positions expected relative to those for the nonplanar distorted geometry for the off state. A 1 ms measurement of the off state corresponds closely with the on-minus-off FTIR difference spectrum, indicating thermal deprotonation and rearrangement of the Arg66 side chain to be complete.

  18. Investigation on the Distribution of Arbovirus in the South of China by Reverse Transcription-Polymerase Chain Reactions%RT-PCR技术在南方虫媒病毒流行病学调查中的应用

    Institute of Scientific and Technical Information of China (English)

    方美玉; 蒋廉华; 陈翠华; 白志军; 林立辉; 刘建伟; 赵文忠; 陈唯军

    2000-01-01

    目的:调查南方地区虫媒病毒感染情况.方法:应用虫媒病毒的通用引物逆转录-聚合酶链反应技术(RT-PCR).结果:检测110组骚扰阿蚊、90组白纹伊蚊和18组致倦库蚊标本,分别检出15组、9组和2组阳性标本,阳性率分别为13.63%、10%、11.11%;168份发烧病人待查标本中,28份疑似登革热血标本、11份疑似乙型脑炎血标本和3份"不明热"血标本,均扩增出413bp的特异带,证实为虫媒病毒感染.结论:①我国南方地区存在除登革病毒和乙型脑炎病毒外的其它黄病毒感染;②3种蚊虫标本检测出的阳性率表明,还可能存在新虫媒病毒疫源地.

  19. 4种动物虫媒病病毒多重RT-PCR检测方法的建立%Simultaneous Detection of 4 Group Arboviruses Using a Single-tube Multiplex Reverse Transcription-Polymerase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    阮周曦; 花群义; 杨俊兴; 曾少灵; 曹琛福; 秦智锋; 吕建强; 林庆燕; 周晓黎

    2010-01-01

    分别设计和合成4对特异性引物,通过对反应条件的优化,初步建立了鉴别蓝舌病病毒(BTV)、鹿流行性出血病病毒(EHDV)、水疱性口炎病毒(VSV)和赤羽病病毒(AKV)的多重RT-PCR检测方法,并对其特异性和敏感性进行了检测.结果表明,所设计的4对引物可对同一样品中的VSV、BTV、EHDV和AKV进行特异性扩增,所扩增的目的片段的长度分别为301、351、537、250 bp.建立的四重RT-PCR检测方法能够检测出10-7稀释的细胞培养病毒液,4种病毒之间没有发生交叉反应,说明该检测方法特异性强,敏感性较高.所建立的多重RT-PCR方法可用于上述4种动物虫媒病病毒的快速鉴别诊断,在动物检疫、临床诊断和流行病学调查方面具有较好的应用前景.

  20. Rapid Detection of Enteroviruses in Water by Improved Reverse Transcription Polymerase Chain Reaction%改进的PCR技术快速检测水中肠道病毒的研究

    Institute of Scientific and Technical Information of China (English)

    袁长青; 叶建锋; 李君文

    2000-01-01

    [目的]快速检测水中肠道病毒.[方法]一步单管反转录PCR,通用引物多重PCR.[结果]反转录与PCR在一个反应管中一步操作即可完成,用6条引物一次反应成功地检测了甲型肝炎病毒(HAV)、脊髓灰质炎病毒(Polio)、柯萨奇B组病毒(CoxB)和埃可病毒(EV).其特异性为半套式PCR所证实,敏感性达101PFU/ml.[结论]一步单管反转录PCR,简化了操作步骤,减少了污染的机会,提高了反应的敏感性和特异性.通用引物多重PCR用较少的引物检测并鉴别较多的病毒,减少了引物之间的干扰,提高了结果的稳定性.

  1. Study on Detecting Transmissible Gastroenteritis Virus by Reverse Transcription Polymerase Chain Reaction%应用RT-PCR检测猪传染性胃肠炎病毒的研究

    Institute of Scientific and Technical Information of China (English)

    朱芬花

    2012-01-01

    根据GenBank已发表的猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)N蛋白的基因序列,设计合成1对引物,扩增片段长度为730 bp,在优化RT-PCR反应条件的基础上,建立了检测TGEV的RT-PCR检测方法,并用该方法对疑似TGEV的猪群临床腹泻疾病进行了诊断检测和分析.本研究建立的TGEV检测方法,特异、灵敏、可靠,为该病的诊断和流行病学调查研究提供了技术保障.%According to N protein gene sequence of TGEV which was published in GenBank, designed and synthesized 1 pair of primers, which amplified fragment length of 730 bp, the detection mthods of TGEV by RT-PCR was established on the basis of the optimization of RT-PCR reaction conditions. This study established the TGEV detection meihods, specific, sensitive and reliable for the diagnosis through clinical detection of diarrheal diseases and which will provide technical support for ep-idemiological studies on transmissible gastroenteritis virus.

  2. Global real-time quantitative reverse transcription-polymerase chain reaction detecting proto-oncogenes associated with 14q32 chromosomal translocation as a valuable marker for predicting survival in multiple myeloma.

    Science.gov (United States)

    Inagaki, Atsushi; Tajima, Emi; Uranishi, Miyuki; Totani, Haruhito; Asao, Yu; Ogura, Hiroka; Masaki, Ayako; Yoshida, Tatsuya; Mori, Fumiko; Ito, Asahi; Yano, Hiroki; Ri, Masaki; Kayukawa, Satoshi; Kataoka, Takae; Kusumoto, Shigeru; Ishida, Takashi; Hayami, Yoshihito; Hanamura, Ichiro; Komatsu, Hirokazu; Inagaki, Hiroshi; Matsuda, Yasufumi; Ueda, Ryuzo; Iida, Shinsuke

    2013-12-01

    CCND1, FGFR3 and c-MAF mRNA expression of tumor samples from 123 multiple myeloma patients were analyzed by global RQ/RT-PCR. CCND1, FGFR3 and c-MAF were positive in 44 (36%), 28 (23%) and 16 (13%) of patients, respectively. In 7 patients, both FGFR3 and c-MAF were positive. The expression of c-MAF was independent unfavorable prognostic factors for overall survival (OS). Autologous stem cell transplantation improved progression-free survival of CCND1-positive patients. Bortezomib, thalidomide or lenalidomide extended OS of FGFR3 and/or c-MAF-positive patients. Thus, CCND1, FGFR3 and c-MAF mRNA expression can predict survival and is useful for planning stratified treatment strategies for myeloma patients.

  3. Development and validation of a pre-column reversed phase liquid chromatographic method with fluorescence detection for the determination of primary phenethylamines in dietary supplements and phytoextracts.

    Science.gov (United States)

    Gatti, Rita; Lotti, Cinzia

    2011-07-15

    A sensitive and selective reversed-phase liquid chromatographic (RP-LC) method was developed and validated to determine octopamine, tyramine and Tyrosine (Tyr) in complex matrices as formulations and phytoextracts (Citrus aurantium), after pre-column derivatization with o-phthaldialdehyde (OPA) reagent. The chromatographic separations were performed at room temperature on a Phenomenex Luna C18 column using methanol and sodium acetate buffer (pH 5.5) by varying composition gradient elution as mobile phase and detected flurometrically at λ(em)=455 nm with λ(ex)=340 nm. The results obtained by the proposed method were compared with those achieved by a validated direct RP-LC method with fluorescence detection at λ(em)=310 nm with λ(ex)=275 nm, as reference method, using a Phenomenex Gemini C18 column under isocratic elution conditions with acetonitrile and sodium 1-heptanesulphonate (pH 3), as mobile phase. The higher sensitivity of the derivatization method (detection limit about 0.06 pmol) allowed the sure determination of octopamine present in traces in the examined samples. The repeatability of method (RSD) was ≤1.90% and there was no significant difference between repeatability and intermediate precision data. Recovery studies showed good results 99.5-101.3% with RSD ranging from 0.8 to 1.2%. All analyses were performed by mild conditions in absence of preliminary difficult extraction methodologies or laborious step of sample pre-treatment.

  4. Simultaneous determination of selected eicosanoids by reversed-phase HPLC method using fluorescence detection and application to rat and human plasma, and rat heart and kidney samples.

    Science.gov (United States)

    Aghazadeh-Habashi, Ali; Asghar, Waheed; Jamali, Fakhreddin

    2015-06-10

    Eicosanoids are biologically active lipid-derived oxidative metabolites of arachidonic acid. We, herein, present an improved sensitive, selective and robust high performance liquid chromatography (HPLC)-fluorescence assay for simultaneous quantification of eicosanoids in human plasma and rat tissues. Aliquots of 200 μL of plasma or 30 mg of heart or kidney tissues were spiked with 16-hydroxydecanoic acid as internal standard, and extracted with anhydrous acetonitrile using solid phase cartridges. The eluted samples were dried, reconstituted in anhydrous acetonitrile and labeled with 2-(2,3-naphthalimino)ethyl-trifluoromethanesulphonate in the presence of saturated potassium fluoride solution in anhydrous acetonitrile and N,N-diiospropylethylamine as catalyst. The derivatized eicosanoids were extracted with anhydrous acetonitrile using solid phase cartridges. Chromatographic separation was achieved on a C18 reversed phase column using gradient mobile phase of 0.05% of formic acid:acetonitrile:water at 0.8 mL/min flow rate. The analytes were detected at excitation and emission wavelength of 260 and 396 nm, respectively. The assay was linear (r(2)≥ 0.98) in the concentration range of 0.01-2.5 μg/mL. The intra-day and inter-day coefficients variation was less than 19.8%. Using this assay, we were able to quantify arachidonic acid metabolites simultaneously in human and rat biological samples.

  5. Organic solvent-free reversed-phase ion-pairing liquid chromatography coupled to atomic fluorescence spectrometry for organoarsenic species determination in several matrices.

    Science.gov (United States)

    Monasterio, Romina P; Londonio, Juan A; Farias, Silvia S; Smichowski, Patricia; Wuilloud, Rodolfo G

    2011-04-27

    A novel method has been developed to determine As-containing animal feed additives including roxarsone (ROX), p-arsanilic acid (p-ASA) and nitarsone (NIT), as well as other organic As species (dimethylarsonic acid (DMAA) and monomethylarsonic acid (MMAA)) by ion-pairing high-performance liquid chromatography coupled to hydride generation atomic fluorescence spectrometry (IP-HPLC-HG-AFS). A simple isocratic reversed-phase (RP) HPLC method with a mobile phase containing citric acid and sodium hexanesulfonate (pH 2.0) was developed using a C(18) column. The use of an organic solvent free mobile phase turns this methodology into an environmentally friendly alternative. Several ion pair forming agents, such as sodium hexanesulfonate, tetrabutylammonium bisulfate and perfluoroheptanoic acid, were studied. The limits of detection for As species were calculated in standard solution and resulted to be 0.2, 0.5, 0.6, 1.6, and 1.6 μg As L(-1) for MMAA, DMAA, p-ASA, ROX and NIT, respectively. This method exhibited convenient operation, high sensitivity and good repeatability. It was applied to As speciation in different samples including arugula, dog food, dog urine and chicken liver.

  6. Gene expression of a green fluorescent protein homolog as a host-specific biomarker of heat stress within a reef-building coral.

    Science.gov (United States)

    Smith-Keune, C; Dove, S

    2008-01-01

    Recent incidences of mass coral bleaching indicate that major reef building corals are increasingly suffering thermal stress associated with climate-related temperature increases. The development of pulse amplitude modulated (PAM) fluorometry has enabled rapid detection of the onset of thermal stress within coral algal symbionts, but sensitive biomarkers of thermal stress specific to the host coral have been slower to emerge. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used to produce fingerprints of gene expression for the reef-building coral Acropora millepora exposed to 33 degrees C. Changes in the expression of 23 out of 399 putative genes occurred within 144 h. Down-regulation of one host-specific gene (AmA1a) occurred within just 6 h. Full-length sequencing revealed the product of this gene to be an all-protein chromatophore (green fluorescent protein [GFP]-homolog). RT-PCR revealed consistent down-regulation of this GFP-homolog for three replicate colonies within 6 h at both 32 degrees C and 33 degrees C but not at lower temperatures. Down-regulation of this host gene preceded significant decreases in the photosynthetic activity of photosystem II (dark-adapted F (v)/F (m)) of algal symbionts as measured by PAM fluorometry. Gene expression of host-specific genes such as GFP-homologs may therefore prove to be highly sensitive indicators for the onset of thermal stress within host coral cells.

  7. Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection.

    Science.gov (United States)

    Xie, Kaizhou; Jia, Longfei; Yao, Yilin; Xu, Dong; Chen, Shuqing; Xie, Xing; Pei, Yan; Bao, Wenbin; Dai, Guojun; Wang, Jinyu; Liu, Zongping

    2011-08-01

    A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.

  8. Quantification of human telomerase reverse transcriptase mRNA in testicular germ cell tumors by quantitative fluorescence real-time RT-PCR.

    Science.gov (United States)

    Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Smith, Gilian L; Newlands, Eward S; Miller, Kurt

    2002-01-01

    Telomerase is a ribonucleoprotein enzyme which is endogenously expressed in germ, stem and tumor cells, but absent in benign somatic cells. The two major telomerase components are human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). It has been shown that hTERT is rate-limiting for telomerase activity and that it plays a central role in human carcinogenesis. Here, we investigated the potential of hTERT and hTR gene expression as diagnostic markers in testicular germ cell tumors (TGCT). hTERT mRNA and hTR expression were quantified in 55 testicular germ cell tumors comprising 36 primary and 19 germ cell tumors from retroperitonal sides by fluorescence real-time RT-PCR using the LightCycler technology. Porphobilinogen deaminase (PBGD) was used as housekeeping gene and to enable relative quantification. For comparison to TGCTs, 38 benign testicular biopsies from patients with fertility disorders were assayed. hTERT expression was detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content (N(hTERT) 38-127). In contrast, mature teratomas from primary and post-chemotherapy masses, which are characterized by well-differentiated tissue components showed a nearly complete downregulation of hTERT expression (N(hTERT) 2-4, pmRNA is expressed in all undifferentiated TGCTs but repressed in mature teratomas. This suggests an inverse correlation between the differentiation status of germ cell tumors and hTERT expression. Thus, detection of hTERT expression in tumors histopathologically classified as mature teratomas enables a molecular-diagnostic confirmation and might aid decision making for treatment of patients presenting with this tumor subtype.

  9. Quantification of human telomerase RNA (Htr) and human telomerase reverse transcriptase (Htert)Mrna in testicular tissue of infertile patients

    Institute of Scientific and Technical Information of China (English)

    Mark Schrader; Markus Miller; Ridiger Heicappell; Bernd Straub; Kurt Miller

    2001-01-01

    Aim: To evaluate the quantitative detection of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients presenting with non-obstructive azoospemia. Methods: hTR and hTERT mRNA expression were quantified in 38 cryopreserved testicular tissue specimens by fluorescence real-time reverse transcription- polymerase chain reaction (RT-PCR)in a LightCycler(r). This was paralleled by conventional histological workup in all tissue specimens and additional semithin sectioning preparation in cases with maturation arrest ( n = 12) and Sertoli-cell-only syndrome ( n = 12). Results: The average normalized hTERT expression (NhTERT) Was 131.9 ± 48.0 copies (mean ± SD) in tissue specimens with full spermatogenesis, NhTERT = 51.2 ± 17.2 copies in those with maturation arrest and NhTERT = 2.7 ± 2.4 copies in those with Sertoli-cell-only syndrome (SCOS). The discriminant analysis showed that detection of NhTERT (NhTR) had a predictive value of 86.8 % (55.3 % ) for correct classification in one of the three histological subgroups.Conclusion: Our results demonstrate that quantitative detection of hTERT mRNA expression in testicular tissue enables a molecular-diagnostic classification of gametogenesis. Quantitative detection of hTERT in testicular biopsies is thus well suited for supplementing the histopathological evaluation.

  10. Cell sorting enables interphase fluorescence in situ hybridization detection of low BCR-ABL1 producing stem cells in chronic myeloid leukaemia patients beyond deep molecular remission.

    Science.gov (United States)

    van Kooten Niekerk, Peter B; Petersen, Charlotte C; Nyvold, Charlotte G; Ommen, Hans B; Roug, Anne S; Nederby, Line; Hokland, Peter; Kjeldsen, Eigil

    2014-01-01

    The exact disease state of chronic myeloid leukaemia (CML) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction (qPCR) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate this, CD34(+) stem cell and progenitor cell subpopulations were isolated by fluorescence-activated cell sorting (FACS), and their content of residual Philadelphia positive (Ph(+) ) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4-log reduction in BCR-ABL1 expression (MR(4) ), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization (iFISH). Despite evaluating fewer cells, iFISH proved superior to mRNA-based qPCR in detecting residual Ph(+) stem cells (P = 0·005), and detected Ph(+) stem- and progenitor cells in 9/10 patients at frequencies of 2-14%. Moreover, while all qPCR(+) samples also were iFISH(+) , 9/33 samples were qPCR-/iFISH(+) , including all positive samples from MR(4) patients. Our findings show that residual Ph(+) cells are low BCR-ABL1 producers, and that DNA-based methods are required to assess the content of persisting Ph(+) stem cells in these patients. This approach demonstrates a clinically applicable manner of assessing residual disease at the stem cell level in CML patients in MR(4) , and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.

  11. Determination of fluorescence-labeled asparaginyl-oligosaccharide in glycoprotein by reversed-phase ultraperformance liquid chromatography with electrospray ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Kurihara, Takamasa; Min, Jun Zhe; Toyo'oka, Toshimasa; Fukushima, Takeshi; Inagaki, Shinsuke

    2007-11-15

    Eight fluorescence reagents, i.e., DBD-F, NBD-F, DNS-Cl, NDA, PSC, FITC, Fmoc-Cl, and DMEQ-COCl, which are reactive to an amino functional group, were tested for the labeling of asparaginyl-oligosaccharides in a glycoprotein. Although the optimal reaction conditions and the fluorescence maximal wavelengths were different for each reagent, the highly sensitive fluorescence detection at the femtomole level of Disialo-Asn (a representative asparaginyl-oligosaccharide) was obtained from the labeling utilizing these reagents. Among them, PSC was the most reliable reagent in terms of detection sensitivity (approximately 3 fmol, signal-to-noise ratio of 5 (S/N = 5) on the chromatogram). However, the structural information could not be obtained from the fluorescence detection. Thus, the on-line determination of a real sample was carried out by UPLC-ESI-TOF-MS. The detection limit of the PSC-labeled Disialo-Asn by selected-ion chromatography was 58 fmol (S/N = 5). When the proposed procedure was applied to the determination of oligosaccharides in ovalbumin, 15 species of PSC-labeled oligosaccharides possessing Man, GlcNAc, and Gal units were identified from the UPLC-ESI-TOF-MS. The number of identified oligosaccharides was relatively greater than the method using Fmoc-Cl. Based on the ovalbumin results, the proposed labeling with PSC followed by UPLC-ESI-TOF-MS detection seems to be useful for the on-line asparaginyl-oligosaccharide analysis.

  12. Application of Reverse Transcription-PCR and Real-Time PCR in Nanotoxicity Research

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2016-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq® DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix. PMID:22975959

  13. Solute and solvent dynamics in confined equal-sized aqueous environments of charged and neutral reverse micelles: a combined dynamic fluorescence and all-atom molecular dynamics simulation study.

    Science.gov (United States)

    Guchhait, Biswajit; Biswas, Ranjit; Ghorai, Pradip K

    2013-03-28

    Here a combined dynamic fluorescence and all-atom molecular dynamics simulation study of aqueous pool-size dependent solvation energy and rotational relaxations of a neutral dipolar solute, C153, trapped in AOT (charged) and IGPAL (neutral) reverse micelles (RMs) at 298 K, is described. RMs in simulations have been represented by a reduced model where SPC/E water molecules interact with a trapped C153 that possesses realistic charge distributions for both ground and excited states. In large aqueous pools, measured average solvation and rotation rates are smaller for the neutral RMs than those in charged ones. Interestingly, while the measured average solvation and rotation rates increase with pool size for the charged RMs, the average rotation rates for the neutral RMs exhibit a reverse dependence. Simulations have qualitatively reproduced this experimental trend and suggested interfacial location for the solute for all cases. The origin for the subnanosecond Stokes shift dynamics has been investigated and solute-interface interaction contribution quantified. Simulated layer-wise translational and rotational diffusions of water molecules re-examine the validity of the core-shell model and provide a resolution to a debate regarding the origin of the subnanosecond solvation component in dynamic Stokes shift measurements with aqueous RMs but not detected in ultrafast IR measurements.

  14. Bioanalysis of bevacizumab and infliximab by high-temperature reversed-phase liquid chromatography with fluorescence detection after immunoaffinity magnetic purification.

    Science.gov (United States)

    Todoroki, Kenichiro; Nakano, Tatsuki; Eda, Yasuhiro; Ohyama, Kaname; Hayashi, Hideki; Tsuji, Daiki; Min, Jun Zhe; Inoue, Koichi; Iwamoto, Naoki; Kawakami, Atsushi; Ueki, Yukitaka; Itoh, Kunihiko; Toyo'oka, Toshimasa

    2016-04-15

    This study presents two simple and rapid methods for the quantification of therapeutic mAbs based on LC. Two mAbs (bevacizumab and infliximab) in plasma samples were purified using magnetic beads immobilized with a commercially-available idiotype antibody for each mAb. Purified mAbs were separated with HT-RPLC and detected with their native fluorescence. Using immunoaffinity beads, each mAb was selectively purified and detected as a single peak in the chromatogram. The HT-RPLC achieved good separation for the mAbs with sharp peaks within 20 min. The calibration curves of the two mAbs ranged from 1 to 20 μg mL(-1) (bevacizumab) and 1-10 μg mL(-1) (infliximab), and they had strong correlation coefficients (r(2) > 0.998). The LOD of bevacizumab and infliximab was 0.07 and 0.15 μg mL(-1), and the LLOQ of bevacizumab and infliximab was 0.12 and 0.25 μg mL(-1), respectively. Thus, the sensitivities were sufficient for clinical analysis. Immunoaffinity purification with HT-RPLC produced a selective and accurate bioanalysis without an LC-MS/MS instrument. Both methods could become general-purpose analytical methods and complement the results obtained with conventional LBA.

  15. [Determination of netilmicin in rat plasma by reversed-phase high performance liquid chromatography with fluorescence detection and pre-column derivatization].

    Science.gov (United States)

    Chang, Xiaojuan; Peng, Jingdong; Liu, Shaopu; Liu, Limin; Dai, Yongkuang

    2009-11-01

    A new, simple and sensitive method based on pre-column derivatization by reversed-phase high performance liquid chromatography (HPLC) is described for the separation and quantification of netilmicin in plasma, using 9-fluorenylmethyl chloroformate (FMOC-Cl) as the derivatization reagent. Its pharmacokinetics is also presented. The derivatization modes and chromatographic conditions were optimized. The separation was performed on an Agilent ZORBAX Eclipse XDB-C8 column (150 mm x 4.6 mm, 5 microm) with a mixture of water-acetonitrile (15:85, v/v) as mobile phase and the flow rate was 1.0 mL/min. The excitation wavelength was 265 nm and the emission wavelength was 315 nm. The linear range was 0.045-8.88 mg/L and the correlation coefficient (r) was 0.9993. The limit of detection (LOD) (S/N = 3) was about 0.01 mg/L, and the limit of quantification was 0.03 mg/L (3LOD) for netilmicin. The relative standard deviation was less than 3% for intra-day assay (n = 5) and 3.5% for inter-day assay (n = 5) and the relative recovery was in the range of 96.62%-100.84% (n = 3). The plasma volume of 30 microL was sufficient for the determination of netilmicin. The method provides a reliable bioanalytical methodology to carry out netilmicin pharmacokinetics in rat plasma.

  16. The reversed terminator of octopine synthase gene on the Agrobacterium Ti plasmid has a weak promoter activity in prokaryotes.

    Science.gov (United States)

    Shao, Jun-Li; Long, Yue-Sheng; Chen, Gu; Xie, Jun; Xu, Zeng-Fu

    2010-06-01

    Agrobacterium tumefaciens transfers DNA from its Ti plasmid to plant host cells. The genes located within the transferred DNA of Ti plasmid including the octopine synthase gene (OCS) are expressed in plant host cells. The 3'-flanking region of OCS gene, known as OCS terminator, is widely used as a transcriptional terminator of the transgenes in plant expression vectors. In this study, we found the reversed OCS terminator (3'-OCS-r) could drive expression of hygromycin phosphotransferase II gene (hpt II) and beta-glucuronidase gene in Escherichia coli, and expression of hpt II in A. tumefaciens. Furthermore, reverse transcription-polymerase chain reaction analysis revealed that an open reading frame (ORF12) that is located downstream to the 3'-OCS-r was transcribed in A. tumefaciens, which overlaps in reverse with the coding region of the OCS gene in octopine Ti plasmid.

  17. Reverse Logistics

    OpenAIRE

    Kulikova, Olga

    2016-01-01

    This thesis was focused on the analysis of the concept of reverse logistics and actual reverse processes which are implemented in mining industry and finding solutions for the optimization of reverse logistics in this sphere. The objective of this paper was the assessment of the development of reverse logistics in mining industry on the example of potash production. The theoretical part was based on reverse logistics and mining waste related literature and provided foundations for further...

  18. Synthesis of mimic molecularly imprinted ordered mesoporous silica adsorbent by thermally reversible semicovalent approach for pipette-tip solid-phase extraction-liquid chromatography fluorescence determination of estradiol in milk.

    Science.gov (United States)

    Wang, Lu; Yan, Hongyuan; Yang, Chunliu; Li, Zan; Qiao, Fengxia

    2016-07-22

    A mimic molecularly imprinted ordered mesoporous silica (MIOMS) adsorbent was prepared utilizing a thermally reversible semicovalent approach. The thermally reversible covalent template-monomer complex was firstly synthesized by employing 4,4'-sulfonyldiphenol (BPS) and (3-isocyanatopropyl) triethoxysilane (ICPTES) as template and monomer, respectively. The template-monomer complex was incorporated into ordered mesoporous silica via a simple self-assembly process. The adsorption experiment illustrated that the imprint-removed silica (MIOMS-ir) had higher special recognition ability (250μgg(-1)) for estradiol (E2) than the non-imprinted silica (NIOMS-ir) (25μgg(-1)). MIOMS-ir was applied as an adsorbent in pipette-tip solid-phase extraction (PT-SPE) coupled with liquid chromatography-fluorescence detector (LC-FLD) for determination of E2 in milk samples. Under the optimized conditions, only 3mg of the adsorbent, 0.3mL of water as washing solvent, and 0.5mL of acetonitrile-acetic acid (96:4, v/v) as elution solvent were used in the pretreatment procedure of milk samples. Good calibration linearity was obtained in a range of 25ngL(-1) to 1000ngL(-1), and the recoveries at three spiked levels were ranged from 95.4% to 107.0% with relative standard deviations (RSDs) ≤3.1% (n=3). The proposed MIOMS-ir-PT-SPE-LC-FLD method combined the advantages of PT-SPE and ordered mesoporous material such as ease assembly, low cost, high extraction efficiency and large specific surface area, so it is a potential pretreatment strategy for the extraction and determination of E2 in complex milk samples.

  19. Failed detection of Bovine viral diarrhea virus 2 subgenotype a (BVDV-2a) by direct fluorescent antibody test on tissue samples due to reduced reactivity of field isolates to raw anti-BVDV antibody.

    Science.gov (United States)

    Yan, Lifang; Pace, Lanny W; Baughman, Brittany; Wilson, Floyd D; Zhang, Shuping; Zhang, Michael Z

    2016-03-01

    Bovine viral diarrhea virus 1 (BVDV-1) is associated with mild or subclinical infections, whereas BVDV-2 is frequently implicated in outbreaks of severe thrombocytopenia and acute fatal disease. In the present study, the carcass of a beef breed cow and tissue samples of a beef calf were received for laboratory diagnosis. Both animals exhibited severe clinical signs compatible with thrombocytopenia or hemorrhagic syndrome. Direct fluorescent antibody test (DFAT) failed to detect BVDV antigen in the tissue specimens of both cases. However, immunohistochemistry (IHC) revealed the presence of BVDV antigen in oral and esophageal mucosa and Peyer patches of the beef breed cow. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) detected BVDV-2 in selected tissues of both animals. Subsequently, BVDV was isolated from both cases and subjected to genetic and serologic characterizations. Mutations in the 5'-untranslated genomic region (5'-UTR) primer and probe binding sites and the E2 gene were associated with reduced efficiency of an established real-time RT-PCR assay and amino acid alterations in the E2 glycoprotein, respectively. Both viral isolates were classified by real-time RT-PCR and phylogenetic analysis as BVDV-2 subgenotype a. Unlike BVDV reference strains Singer and 125c, the isolates cross-reacted with anti-BVDV-1 and anti-BVDV-2 reference sera, indicating antigenic variations in field isolates. The isolates also showed reduced reactivity to porcine anti-BVDV antiserum (the raw serum used to produce BVDV DFA conjugate). In summary, data from the present investigation indicated that genetic and antigenic variations affected the performance of detection assays, especially DFAT, highlighting the need for regular evaluation and modification of BVDV tests.

  20. Simultaneous determination of 11 antibiotics and their main metabolites from four different groups by reversed-phase high-performance liquid chromatography-diode array-fluorescence (HPLC-DAD-FLD) in human urine samples.

    Science.gov (United States)

    Fernandez-Torres, R; Consentino, M Olías; Lopez, M A Bello; Mochon, M Callejon

    2010-05-15

    A new, accurate and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) as analytical method for the quantitative determination of 11 antibiotics (drugs) and the main metabolites of five of them present in human urine has been worked out, optimized and validated. The analytes belong to four different groups of antibiotics (sulfonamides, tetracyclines, penicillins and anphenicols). The analyzed compounds were sulfadiazine (SDI) and its N(4)-acetylsulfadiazine (NDI) metabolite, sulfamethazine (SMZ) and its N(4)-acetylsulfamethazine (NMZ), sulfamerazine (SMR) and its N(4)-acetylsulfamerazine (NMR), sulfamethoxazole (SMX), trimetroprim (TMP), amoxicillin (AMX) and its main metabolite amoxicilloic acid (AMA), ampicillin (AMP) and its main metabolite ampicilloic acid (APA), chloramphenicol (CLF), thiamphenicol (TIF), oxytetracycline (OXT) and chlortetracycline (CLT). For HPLC analysis, diode array (DAD) and fluorescence (FLD) detectors were used. The separation of the analyzed compounds was conducted by means of a Phenomenex Gemini C(18) (150mm x 4.6mm I.D., particle size 5microm) analytical column with LiChroCART LiChrospher C(18) (4mm x 4mm, particle size 5microm) guard column. Analyzed drugs were determined within 34min using formic acid 0.1% in water and acetonitrile in gradient elution mode as mobile phase. A linear response was observed for all compounds in the range of concentration studied. Two procedures were optimized for sample preparation: a direct treatment with methanol and acetonitrile and a solid phase extraction procedure using Bond Elut Plexa columns. The method was applied to the determination of the analytes in human urine from volunteers under treatment with different pharmaceutical formulations. This method can be successfully applied to routine determination of all these drugs in human urine samples.

  1. Fluorescence spectroscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2016-01-01

    Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses...

  2. Photophysical properties of pyronin dyes in reverse micelles of AOT

    Energy Technology Data Exchange (ETDEWEB)

    Bayraktutan, Tuğba; Meral, Kadem; Onganer, Yavuz, E-mail: yonganer@atauni.edu.tr

    2014-01-15

    The photophysical properties of pyronin B (PyB) and pyronin Y (PyY) in reverse micelles formed with water/sodium bis (2-ethyl-1-hexyl) sulfosuccinate (AOT)/n-heptane were investigated by UV–vis absorption, steady-state and time-resolved fluorescence spectroscopy techniques. This study was carried out a wide range of reverse micelle sizes, with hydrodynamic radii ranging from 1.85 to 9.38 nm. Significant photophysical parameters as band shifts, fluorescence quantum yields and fluorescence lifetimes were determined to understand how photophysical and spectroscopic features of the dye compounds were affected by the variation of reverse micelle sizes. In this regard, control of reverse micelle size by changing W{sub 0}, the molar ratio of water to surfactant, allowed tuning the photophysical properties of the dyes in organic solvent via reverse micelle. Non-fluorescent H-aggregates of pyronin dyes were observed for the smaller reverse micelles whereas an increase in the reverse micelle size induced an increment in the amount of dye monomers instead of dye aggregates. Thus, the fluorescence intensities of the dyes were improved by increasing W{sub 0} due to the predomination of the fluorescent dye monomers. As a result, the fluorescence quantum yields also increased. The fluorescence lifetimes of the dyes in the reverse micelles were determined by the time-resolved fluorescence decay studies. Evaluation of the fluorescence lifetimes calculated for pyronin dyes in the reverse micelles showed that the size of reverse micelle affected the fluorescence lifetimes of pyronin dyes. -- Highlights: • The photophysical properties of pyronin dyes were examined by spectroscopic techniques. • Optical properties of the dyes were tuned by changing of W{sub 0} values. • The fluorescence lifetime and quantum yield values of the dyes in reverse micelles were discussed.

  3. Reverse logistics

    NARCIS (Netherlands)

    M.P. de Brito (Marisa); S.D.P. Flapper; R. Dekker (Rommert)

    2002-01-01

    textabstractThis paper gives an overview of scientific literature that describes and discusses cases of reverse logistics activities in practice. Over sixty case studies are considered. Based on these studies we are able to indicate critical factors for the practice of reverse logistics. In addi

  4. Constructing Competitive Reverse Transcription Polymerize Chain Reaction Inter-Reference of PC mRNA by Intron Approach

    Institute of Scientific and Technical Information of China (English)

    XU Chuang; XIA Cheng; LIU Guo-wen; WANG Zhe; JIANG Yu-fu; ZHANG Nai-sheng; FU Shi-xin

    2004-01-01

    Inter-reference ofcompetitive reverse transcription polymerase chain reaction(RT-PCR)was constructed by intron method to detect the change of PC mRNA level in the pathway of carbohydrate metabolism.The experiment based on the principle that 81bp intron sequence was deleted in PC mRNA compared with PC DNA sequence.The 466bp competitive DNA template recombinant plasmid of PC mRNAwas successfully built by a pair of primer and was cloned once,PC DNA and PC mRNA could be inter-referred each other.The intron approach used in the experiment has broken through the traditional method of constructing competitive template.

  5. LNA探针同时检测人副流感病毒1,2,3型多重荧光定量RT-PCR方法的建立%Simultaneous detection of human parainfluenza viruses 1, 2, 3 by multiplex real-time reverse transcription-polymerase chain reaction with LNA probes

    Institute of Scientific and Technical Information of China (English)

    姬奕昕; 毛乃颖; 王焕焕; 谢正德; 许文波

    2012-01-01

    目的 人副流感病毒1,2,3型是呼吸道感染的主要病原.本研究建立了特异、快速、灵敏的多重荧光定量RT-PCR方法用于人副流感1,2,3型病毒临床标本检测.方法 针对人副流感1,2,3型病毒设计特异性引物探针,优化荧光RT-PCR反应条件.应用体外转录方法分别制备人副流感1,2,3型病毒的标准品.验证荧光定量RT-PCR方法的特异性,敏感性和稳定性.结果 该方法对人副流感1,2,3型病毒核酸检测有高度特异性,检测的灵敏度HPIV1为10个拷贝,HPIV2为100个拷贝,HPIV3为100个拷贝.可从临床患者鼻咽吸出物标本中直接检出.结论 本研究建立的LNA探针同时检测人副流感病毒1,2,3型多重荧光定量RT-PCR方法具有较高的特异性和敏感性.适用于临床早期诊断和实验室病原谱筛查.%Objective Human parainfluenza virus (HPIV) types 1,2 and 3 are major viral pathogens responsible for upper and lower respiratory tract infections.In this study,a real-time RT-PCR was developed using multiplex primers-probe (HPIV-1,2,3) for the simultaneous detection of both HPIV1,HPIV2 and HPIV3 genomes.Methods Optimal primers and probes were designed using specialized software.The conditions for multiplex real-time RT-PCR had been optimized.The synthesis of RNA standards of HPIV1,2,3 were used a T7 RNA polymerase.Check the specificity sensitivities and stability of one step RT-PCR assay.Results Obtained in a 10-fold dilution series assay demonstrate a high sensitivity of the assay with a lowest detection limit of 10 copies for HPIV1,100 copies for HPIV2 and 100 copies for HPIV3.Conclusion The assays demonstrates an improved sensitivity and scope of detecting HPIV1,2,3 viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the pre-diagnosis and respiratory virus pathogenesis.

  6. 应用巢式逆转录聚合酶链反应检测鱼类病毒性出血性败血症病毒(VHSV)%DETECTION OF FISH VIRAL HEMORRHAGIC SEPTICEMIA VIRUS (VHSV) BY NESTED REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR)

    Institute of Scientific and Technical Information of China (English)

    倪穗; 余晓巍; 王建平; 雷爱莹; 曾地刚; 吴雄飞

    2009-01-01

    参照鱼类病毒性出血性败血症病毒序列,根据PCR引物设计的原则,设计巢式PCR引物,采用巢式逆转录聚合酶链反应(RT-PCR)方法,对如何快速检测鱼类病毒性出血性败血症病的方法进行了较为系统的研究,并对RT-PCR的反应条件进行了优化.结果表明,巢式RT-PCR扩增获得279bp的特异性片断,阴性对照无扩增条带.巢式RT-PCR扩增出的特异性片段经测序分析,结果证实与报道的序列完全一致.该方法最低可检测出0.1pg的鱼类病毒性出血性败血症病毒RNA.初步建立了VHSV的巢式RT-PCR检测方法,该方法灵敏、特异,可为VHSV的检测提供一个快速、有效的手段.

  7. Differential detection of porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus with reverse transcription-polymerase chain reaction%猪流行性腹泻病毒和猪传染性胃肠炎病毒的RT-PCR鉴别诊断

    Institute of Scientific and Technical Information of China (English)

    方立; 徐辉; 陈伟杰; 赵灵燕; 倪柏锋; 方维焕

    2006-01-01

    猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)为套式病毒目冠状病毒科、冠状病毒属的病毒,引起猪流行性腹泻(porcine epidemic diarrhea,PED)。该病是一种猪肠道传染病,以水泻、呕吐和脱水为特征,各种年龄的猪均易感染,哺乳仔猪、架子猪或育肥猪的发病率可达100%,尤其是哺乳仔猪受害最严重,该病主要发生于冬季,夏季也可发生。猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)为套式病毒目冠状病毒科、冠状病毒属的病毒,是猪传染性胃肠炎(porcine transmissible gastroenteritis,TGE)的主要病原。该病是一种急性、高度接触性传染病,以呕吐、严重腹泻、脱水为特征的传染病,对新生仔猪具有高度致死率。虽然不同年龄的猪对这种病毒均易感,但5周龄以上的猪死亡率较低,多成僵猪,使养猪饲料报酬降低。

  8. Reversible Computing

    Science.gov (United States)

    1980-02-01

    will have been introduced. 9. Reversible celular autemata We shall assume the reader to have some familiarity with the concept of cel- lular...10003 Mr. Kin B. Thcmpson 1 copy Technical Director Information Systems Divisia.i Naval Research Laboratory (OP-91T) Technical Information Division

  9. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  10. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  11. Diagnostic accuracy of FISH and RT-PCR in 50 routinely processed synovial sarcomas

    NARCIS (Netherlands)

    Ten Heuvel, Suzan E.; Hoekstra, Harald J.; Suurmeijer, Albert J. H.

    Background: Molecular detection of SYT-SSX fusion genes is the most reliable tool for diagnosing synovial sarcoma (SS). The objective of this study was to investigate the accuracy of reverse transcription-polymerase chain reaction (RT-PCR) and a commercially available fluorescence in situ

  12. Reversible Histone Acetylation Involved in Transcriptional Regulation of WT1 Gene

    Institute of Scientific and Technical Information of China (English)

    Yangguang SHAO; Jun LU; Cao CHENG; Liguo CUI; Guoping ZHANG; Baiqu HUANG

    2007-01-01

    To validate the involvement of reversible histone acetylation in the transcriptional regulation of human Wilms' tumor 1 gene (WT1), we analyzed the roles of histone deacetylases (HDACs) and histone acetyltransferase in this epigenetic process. Of the six HDACs (HDAC1-6) examined, HDAC4 and HDAC5 were found to have significant repressing effects on the activity of the WT1 reporter gene, as revealed by luciferase reporter assays and quantitative real-time reverse transcription-polymerase chain reaction assays.Luciferase reporter assays showed that the histone acetyltransferase p300 was able to counteract the HDAC4/HDAC5-mediated repression and that p300/CBP synergized with transcription factors Sp1, c-Myb, and Ets-1 in activation of the WT1 reporter. Chromatin immunoprecipitation experiments showed that p300 promotes the acetylation level of histone H3 at the WT1 intronic enhancer. Based on these data, we proposed a hypothetical model for the involvement of reversible histone acetylation in transcriptional regulation of the WT1 gene. This study provides further insight into the mechanisms of transcriptional regulation of the WT1 gene and WT1-associated diseases treatment.

  13. Synthesis and Application of Four Fluorescence Labeled Nucleotides Through Disulfide as Reversible Terminators in DNA Sequencing by Synthesis%四色荧光标记二硫键可逆终端的合成及在DNA合成测序中的应用

    Institute of Scientific and Technical Information of China (English)

    汤道年; 江敏; 李小卫; 康亚妮; 伍新燕; 龚兵; 邵志峰; 赵小东; 沈玉梅

    2014-01-01

    分别以5-碘-2'-脱氧尿苷、5-碘-2'-脱氧胞苷、7-去氮-7-碘-2'-脱氧腺苷及7-去氮-7-碘-2'-脱氧鸟苷为原料,以二硫键为可裂解连接单元,通过多步反应合成了四色荧光标记不同碱基的脱氧核糖核苷酸;研究了该类荧光标记核苷酸作为可逆终端在DNA合成测序中的应用。所得产物结构经1 H NMR,31 P NMR及HRMS表征,并对其进行了DNA高通量测序的测试。结果表明,该类荧光标记核苷酸作为DNA合成测序的可逆终端能够满足高通量测序的生化反应要求,具有较好的应用前景。%DNA sequencing is one of the important means of biological research. The development of accurate and high-throughput DNA-sequencing methods has a strong impact on biomedical research. Sequencing by synthesis( SBS) approaches is one of the most important techniques of DNA sequencing. A series of SBS methods have been investigated and implemented. In SBS methods, the principal thing is to synthesize four fluorescently labelled cyclic reversible terminators as the sequencing reagents. These cyclic reversible termina-tors need to connect the fluorescein through a cleavable linker. In chemical biology, disulfide bridges, which are known to be efficiently and rapidly cleaved by mild reducing agents, have been used in a wide range of ap-plications including drug delivery, functional and structural proteomics, DNA and tumor imaging. The disul-fide-based cleavable linker is commonly used due to its rapid cleavage and straightforward synthesis. In this work, starting from 5-iodo-2'-deoxyuridine, 5-iodo-2'-deoxycyridine, 7-deazo-7-iodo-2'-deoxyadenosine and 7-deazo-7-iodo-2'-deoxyguanosine, four kinds of fluorescence labelled necleotides with four different bases through disulfide were synthesized and characterized. The synthesized fluorescence labelled nucleotides can be recognized and accepted by an identified DNA polymerase, incorporated faithfully into DNA strand, followed by

  14. Reversible Statistics

    DEFF Research Database (Denmark)

    Tryggestad, Kjell

    2004-01-01

    The study aims is to describe how the inclusion and exclusion of materials and calculative devices construct the boundaries and distinctions between statistical facts and artifacts in economics. My methodological approach is inspired by John Graunt's (1667) Political arithmetic and more recent work...... within constructivism and the field of Science and Technology Studies (STS). The result of this approach is here termed reversible statistics, reconstructing the findings of a statistical study within economics in three different ways. It is argued that all three accounts are quite normal, albeit...... in different ways. The presence and absence of diverse materials, both natural and political, is what distinguishes them from each other. Arguments are presented for a more symmetric relation between the scientific statistical text and the reader. I will argue that a more symmetric relation can be achieved...

  15. Multichromophoric sugar for fluorescence photoswitching

    Directory of Open Access Journals (Sweden)

    Stéphane Maisonneuve

    2014-06-01

    Full Text Available A multichromophoric glucopyranoside 2 bearing three dicyanomethylenepyran (DCM fluorophores and one diarylethene (DAE photochrome has been prepared by Cu(I-catalyzed alkyne–azide cycloaddition reaction. The fluorescence of 2 was switched off upon UV irradiation, in proportion with the open to closed form (OF to CF conversion extent of the DAE moiety. A nearly 100% Förster-type resonance energy transfer (FRET from all three DCM moieties to a single DAE (in its CF moiety was achieved. Upon visible irradiation, the initial fluorescence intensity was recovered. The observed photoswiching is reversible, with excellent photo resistance.

  16. Fluorescent microspheres

    Science.gov (United States)

    Rembaum, A.

    1978-01-01

    Latex particles with attached antibodies have potential biochemical and environmental applications. Human red blood cells and lymphocytes have been labeled with fluorescent microspheres by either direct or indirect immunological technique. Immunolatex spheres can also be used for detecting and localizing specific cell surface receptors. Hormones and toxins may also be bondable.

  17. Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification.

    Science.gov (United States)

    Silva, Gonçalo; Bömer, Moritz; Nkere, Chukwuemeka; Kumar, P Lava; Seal, Susan E

    2015-09-15

    Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam production globally. Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential component of disease control. Current serological and PCR-based diagnostic methods for YMV are time consuming involving a succession of target detection steps. In this study, a novel assay for specific YMV detection is described that is based on isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown to be reproducible and able to detect as little as 14 pg/μl of purified RNA obtained from an YMV-infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay a promising candidate for adapting into a field test format to be used by yam breeding programmes or certification laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Large GLUT4 vesicles are stationary while locally and reversibly depleted during transient insulin stimulation of skeletal muscle of living mice: imaging analysis of GLUT4-enhanced green fluorescent protein vesicle dynamics

    DEFF Research Database (Denmark)

    Lauritzen, Hans P M M; Galbo, Henrik; Brandauer, Josef

    2007-01-01

    OBJECTIVE: Insulin stimulates glucose transport in skeletal muscle by GLUT4 translocation from intracellular compartments to sarcolemma and t-tubules. We studied in living animals the recruitment of GLUT4 vesicles in more detail than previously done and, for the first time, analyzed the steady......-state recycling and subsequent re-internalization of GLUT4 on an insulin bolus. RESEARCH DESIGN AND METHODS: A confocal imaging technique was used in GLUT4-enhanced green fluorescent protein-transfected superficial muscle fibers in living mice. RESULTS: During the first 30 min of insulin stimulation, very few...... superficially or deeply located GLUT4 storage vesicles (>1 microm) moved in toto. Rather, big vesicles were stationary in their original position at sarcolemma or t-tubules and were locally depleted of GLUT4 by budding off of smaller vesicles. Photobleaching experiments revealed that during initial...

  19. Effects of fluorescent light and vacuum packaging on the rate of decomposition of pigments in paprika (Capsicum annuum) powder determined by reversed-phase high-performance liquid chromatography.

    Science.gov (United States)

    Morais, H; Ramos, A C; Tibor, C; Forgács, E

    2001-11-30

    The effect of storage time, the presence of light and oxygen on the decomposition rate of carotenoid pigments in paprika (Capsicum annuum) powders was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). The similarities and dissimilarities of pigment composition of samples under various storage conditions was elucidated by principal component analysis (PCA) and stepwise regression analysis (SRA). Calculations proved that the overall decomposition rate of pigment sections equally depended on the storage time and on the presence of light and oxygen, the effect of storage time being the most decisive factor while the impact of oxygen was the lowest. The selectivity of decomposition also depended on the storage time and on the presence of oxygen the influence of storage time being the most important. RP-HPLC followed by PCA and SRA can be successfully used for the study of the impact of environmental conditions on the decomposition of carotenoid pigments of paprika powders.

  20. Rapid detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Li, Lin; Bao, Jingyue; Wu, Xiaodong; Wang, Zhiliang; Wang, Junwei; Gong, Mingxia; Liu, Chunju; Li, Jinming

    2010-12-01

    Peste des petits ruminants virus (PPRV) is the causative agent of peste des petits ruminants (PPR), an economically important viral disease of small ruminants. In this report, a one-step, single-tube, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PPRV. A set of six LAMP primers were designed based on the matrix gene sequence of PPRV to amplify the target RNA by incubation at 63°C for 60min with Bst DNA polymerase and reverse transcriptase. The amplified products could be observed by the naked eye. The specificity of the RT-LAMP assay was validated by amplifying eight strains of PPRV isolated in different geographical areas. No cross-reactivity with other related viruses, including rinderpest virus, canine distemper virus and measles virus, was detected. The sensitivity of the assay was similar to that of real-time reverse transcription polymerase chain reaction (RT-PCR) and 10-fold higher than that of conventional RT-PCR. Twenty clinical samples were evaluated by the RT-LAMP assay, and the results were consistent with those of real-time RT-PCR. As a simple, rapid and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of PPR and the surveillance of PPRV. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. Stathmin is key in reversion of doxorubicin resistance by arsenic trioxide in osteosarcoma cells.

    Science.gov (United States)

    Feng, Tao; Qiao, Guanglei; Feng, Li; Qi, Weixiang; Huang, Yujing; Yao, Yang; Shen, Zan

    2014-12-01

    Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. Numerous patients are unable to be cured due to the development of resistance of the osteosarcoma cells to chemotherapeutic drugs. Therefore, reversion of drug resistance is urgently required for the treatment of osteosarcoma. Arsenic trioxide (As2O3) is an active ingredient in Traditional Chinese Medicine, but the therapeutic potential of As2O3 in osteosarcoma remains largely unexplored. The current study investigated the effects of As2O3 on MG63 osteosarcoma cells using a cell proliferation assay, flow cytometric analysis of the cell cycle and cell apoptosis, reverse transcription polymerase chain reaction to detect stathmin mRNA expression levels and western blot analysis to detect the stathmin protein expression levels. As2O3 and doxorubicin (ADM) combination treatment markedly inhibited cell proliferation in ADM-resistant MG63 (MG63/dox) osteosarcoma cells, clearly induced G2/M phase cell cycle arrest and increased the number of apoptotic MG63/dox cells. Furthermore, stathmin expression was found to be downregulated in MG63/dox cells and was sensitive to ADM treatment. Additional investigation revealed that the downregulation of stathmin expression in MG63/dox cells by stathmin small interfering RNA significantly enhanced the reversion of ADM resistance in MG63/dox by As2O3. The data indicated that As2O3 reversed ADM resistance in MG63/dox cells through downregulation of stathmin and may be a potential drug for the treatment of ADM-resistant osteosarcoma.

  2. Absorption Complex between Porphyrin and Phenothiazine in Reverse Micelles

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The interaction between amphiphilic porphyrin and phenothiazine in AOT/isooctane/ water reverse micelle was investigated by UV-Vis spectra. A new absorption complex between the two species is formed in such circumstances, which is ascribed to the enrichment of the components by the reverse micelle. The fluorescence quenching of CHTTP by PTH becomes more efficient after the formation of the absorption complex.

  3. Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

    Directory of Open Access Journals (Sweden)

    Ravi Prakash

    2014-12-01

    Full Text Available Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR is facilitated by leveraging droplet microfluidic (DMF system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points. This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs. The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind panels of extracted patient samples with acceptably high PCR efficiency (>94%. The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.

  4. 应用间期荧光原位杂交技术检测慢性髓系白血病患者的肿瘤负荷%Application of Interphase Fluorescence In Situ Hybridization for Determination of the Tumor Load in Chronic Myeloid Leukemia

    Institute of Scientific and Technical Information of China (English)

    杜庆锋; 周淑芸; 刘晓力; 宋兰林; 张钰

    2003-01-01

    背景与目的:在评价慢性髓系白血病 (chronic myeloid leukemia,CML)对各种治疗如 IFN-α、骨髓移植等的反应程度时,准确判断患者骨髓细胞中的肿瘤负荷十分重要.本研究旨在探讨间期荧光原位杂交 (interphase fluorescence in situ hybridization,I- FISH)技术监测 CML患者治疗过程中肿瘤负荷的灵敏度与特异性.方法:应用 I- FISH技术对 10例对照的骨髓细胞进行检测并确定正常分界值 ;对 20例治疗中 CML患者骨髓中的肿瘤负荷进行检测 ,并将结果与常规细胞遗传学 (conventional cytogenetics,CCG)G显带及筑巢式逆转录聚合酶链式反应 (reverse transcription- polymerase chain reaction,RT- PCR)技术检测的结果进行比较.结果: I- FISH检测对照组骨髓细胞阳性细胞率为 0 6%~ 2 0% ,分界值为 2 45% ;20例 CML患者经 IFN-α治疗或骨髓移植后, 9例经 CCG检测到 Ph(+ )细胞 ,阳性细胞率 16 7%~ 100% ;结合正常分界值 ,20例中有 16例 (80% )经 I- FISH检测到 bcr- abl(+ )肿瘤细胞 ,阳性细胞率 2 8%~ 99 6%.这 16例患者行 RT- PCR检查,其中 13例 (81 3% )融合基因转录本阳性.结论: I- FISH技术可较灵敏、特异地监测 CML患者治疗过程中肿瘤细胞负荷的变化.

  5. Managing Reverse Logistics or Reversing Logistics Management?

    OpenAIRE

    Brito, Marisa

    2004-01-01

    textabstractIn the past, supply chains were busy fine-tuning the logistics from raw material to the end customer. Today an increasing flow of products is going back in the chain. Thus, companies have to manage reverse logistics as well.This thesis contributes to a better understanding of reverse logistics. The thesis brings insights on reverse logistics decision-making and it lays down theoretical principles for reverse logistics as a research field.In particular it puts together a framework ...

  6. Study on the Interaction between CdTe Quantum Dot-Acridine Orange-Calf Thymus DNA by Fluorescence Reversible Control%荧光可逆调控研究CdTe量子点-吖啶橙-小牛胸腺DNA的相互作用及分析应用

    Institute of Scientific and Technical Information of China (English)

    龚会平; 刘绍璞; 殷鹏飞; 闫曙光; 范小青; 何佑秋

    2011-01-01

    水相合成了谷胱甘肽(GSH)修饰的CdTe量子点(QDs).在PH=7.4的Tris-HCl缓冲溶液中,吖啶橙(AO)通过静电引力吸附到GSH-CdTe QDs的表面,与GSH-CdTe QDs形成了基态复合物,导致GSH-CdTe QDs的荧光猝灭.在GSH-CdTe QDs-AO体系中加入小牛胸腺DNA(ctDNA),ctDNA诱导AO从GSH-CdTe QDs表面脱落嵌入其双螺旋结构中,导致GSH-CdTe QDs的荧光恢复.根据GSH-CdTe QDs荧光的猝灭和恢复,实现了量子点荧光的可逆调控.ctDNA引起GSH-CdTe QDs-AO体系荧光恢复强度与ctDNA浓度成良好的线性关系,检出限为0.13 ng mL-1,据此提出了简便快捷、准确、高灵敏测定ctDNA的新方法.还结合共振瑞利散射(RRS)光谱、吸收光谱和原子力显微镜照片研究了GSH-CdTe QDs-AO-ctDNA三者之间的相互作用,对相互作用机理进行了讨论并提出了相应的作用模型.%Glutathione(GSH)-capped CdTe quantum dots(GSH-CdTe QDs) were synthesized in aqueous solution.In pH 7.4 Tris-HCl buffer medium,acridine orange(AO) was adsorbed to the surfaces of GSH-CdTe QDs via electrostatic attraction and formed ground state complex,which resulted in the quenching of the fluorescence of GSH-CdTe QDs.Adding ctDNA to GSH-CdTe QDs-AO system leaded to the fluorescence intensity of GSH-CdTe QDs recover,which can be explained by that the addition of ctDNA to the system induced AO to dissociate from the surface of GSH-CdTe QDs and embed into its double helix structure.According to the fluorescence quencher and restoration for GSH-CdTe QDs,fluorescence reversible control of QDs was realized.The fluorescence intensity change of GSH-CdTe QDs-AO system aroused by the addition of ctDNA was proportional to the ctDNA concentration in a certain range,and its detection limit was 0.13 ngomL-1.Based on it,the simple,rapid,accurate and sensitive methods had been proposed to determine ctDNA.The interaction of GSH-CdTe QDs-AO-ctDNA was studied by resonance Rayleigh scattering

  7. Reverse Genetic Analysis of Transcription FactorOsHox9, a Member of Homeobox Family, in Rice

    Institute of Scientific and Technical Information of China (English)

    AI Li-ping; SHEN Ao; GAO Zhi-chao; LI Zheng-long; SUN Qiong-lin; LI Ying-ying; LUAN Wei-jiang

    2014-01-01

    Homeobox transcription factors participate in the growth and development of plants by regulating cell differentiation, morphogenesis and environmental signal response. To reveal the functions of these transcription factors in rice, we constructed the RNAi vectors ofOsHox9, a member of homeobox family, and analyzed the function ofOsHox9 using reverse genetics. The plant height and tillering number of RNAi transgenic plants decreased compared with those of wild-type plants. Reverse transcription-polymerase chain reaction analysis showed thatOsHox9 expression reduced in the transgenic plants with phenotypic variance, whereas that in the transgenic plants without phenotypic variance was similar to that in the wild-type plants. This result suggests that the phenotypes of the transgenic plants were caused by RNAi effects. The tissue-specificity ofOsHox9 expression indicated that it was expressed in different organs, with high expression in stem apical meristem and young panicles. Subcelular location ofOsHox9 demonstrated that it was localized on the cell membrane.

  8. Testicular expression of survivin and human telomerase reverse transcriptase (hTERT) associated with spermatogenic function in infertile patients

    Institute of Scientific and Technical Information of China (English)

    Steffen Weikert; Frank Christoph; Wolfgang Schulze; Hans Krause; Carsten Kempkensteffen; Martin Schostak; Kurt Miller; Mark Schrader

    2006-01-01

    Aim: To characterize the coexpression of survivin, an inhibitor of apoptosis (IAF), and human telomerase reverse transcriptase (hTERT) in human testes with varying spermatogenic function. Methods: Transcript levels of survivin mRNA and hTERT mRNA were determined in normal testes (n = 11) and testes with defective spermatogenesis (n = 28) using real-time reverse-transcription polymerase chain reaction (RT-PCR). The histological work-up was performed according to a modified Johnsen score. Results: Expressions of both survivin and hTERT were highest at median levels of 96.8 and 709 in normal spermatogenesis and dropped to 53.3 and 534 in testes with postmeiotic spermatogenic arrest (n = 10). In severe spermatogenic failure (n = 18), survivin expression was lacking in most specimens (n = 16), whereas at least low levels of testicular hTERT expression were largely detectable with a normalized expression of 73 in premeiotic spermatogenic arrest (n = 7) and 45 in patients with Sertoli cell-only syndrome (SCOS)(n = 3). Both survivin and hTERT expressions increased with a progressing Johnsen score (P for trend = 0.001).Conclusion: Although both survivin and hTERT are correlated with spermatogenic function, they show different expression patterns in testes of infertile patients. These findings substantiate results from studies in the rodent testis suggesting a predominant expression of survivin in meiotically dividing germ cells.

  9. Managing Reverse Logistics or Reversing Logistics Management?

    NARCIS (Netherlands)

    M.P. de Brito (Marisa)

    2004-01-01

    textabstractIn the past, supply chains were busy fine-tuning the logistics from raw material to the end customer. Today an increasing flow of products is going back in the chain. Thus, companies have to manage reverse logistics as well.This thesis contributes to a better understanding of reverse

  10. Managing Reverse Logistics or Reversing Logistics Management?

    NARCIS (Netherlands)

    M.P. de Brito (Marisa)

    2004-01-01

    textabstractIn the past, supply chains were busy fine-tuning the logistics from raw material to the end customer. Today an increasing flow of products is going back in the chain. Thus, companies have to manage reverse logistics as well.This thesis contributes to a better understanding of reverse log

  11. Reversible Thermoset Adhesives

    Science.gov (United States)

    Mac Murray, Benjamin C. (Inventor); Tong, Tat H. (Inventor); Hreha, Richard D. (Inventor)

    2016-01-01

    Embodiments of a reversible thermoset adhesive formed by incorporating thermally-reversible cross-linking units and a method for making the reversible thermoset adhesive are provided. One approach to formulating reversible thermoset adhesives includes incorporating dienes, such as furans, and dienophiles, such as maleimides, into a polymer network as reversible covalent cross-links using Diels Alder cross-link formation between the diene and dienophile. The chemical components may be selected based on their compatibility with adhesive chemistry as well as their ability to undergo controlled, reversible cross-linking chemistry.

  12. Velocity selective optical pumping resonance sign reversal

    Science.gov (United States)

    Krasteva, A.; Slavov, D.; Todorov, G.; Cartaleva, S.

    2013-03-01

    We report experimental and theoretical examinations of the peculiarities in Velocity Selective Optical Pumping (VSOP) resonance behavior at open and closed hyperfine transition spectra of Cs atoms (on the D2 line), confined in optical cell with thickness L = 6λ, where λ = 852 nm. For linear and circular polarizations of the irradiating light, open transitions exhibit reduced absorption (fluorescence) VSOP resonances whose contrast increases with atomic concentration and light intensity. However, in case of closed transition the situation is different, the enhanced absorption (fluorescence) VSOP resonance reverses its sign with the atomic concentration and light intensity. Theoretical analysis based on the density matrix formalism, taking into account the statistical tensors describing atomic population and longitudinal alignment, shows that the VSOP resonance sign reversal at the closed transition can be attributed to the efficiency reduction of population transfer by the spontaneous decay with atomic source temperature.

  13. Insight into a reversible energy transfer system.

    Science.gov (United States)

    Gao, Ming Xuan; Zou, Hong Yan; Gao, Peng Fei; Liu, Yue; Li, Na; Li, Yuan Fang; Huang, Cheng Zhi

    2016-09-15

    Resonance energy transfer (RET) processes have wide applications; these processes involve a unidirectional energy transfer from a particular donor to a particular acceptor. Here, we report a plasmonic resonance energy transfer (PRET), which occurs from the surface of gold nanoparticles to fluorescent organic dyes, and coexists with a nanometal surface energy transfer (NSET) that operates in the reverse direction. The coexistence of both PRET and NSET in opposite directions means that the roles of both donor and acceptor can be interchanged, which could be identified by using spectrofluorometric measurements and light scattering dark field microscopic imaging. The experimental data could be further theoretically supported using Persson and Lang's model, the quasi-static approximation and finite-difference time-domain simulation. Moreover, disruption of the PRET process by altering the energy transfer pairs suggests that interactions occur inside the reversible energy transfer system, which manifest by increasing the fluorescence quenching efficiency of the NSET process.

  14. Fluorescence enhancement of photoswitchable metal ion sensors

    Science.gov (United States)

    Sylvia, Georgina; Heng, Sabrina; Abell, Andrew D.

    2016-12-01

    Spiropyran-based fluorescence sensors are an ideal target for intracellular metal ion sensing, due to their biocompatibility, red emission frequency and photo-controlled reversible analyte binding for continuous signal monitoring. However, increasing the brightness of spiropyran-based sensors would extend their sensing capability for live-cell imaging. In this work we look to enhance the fluorescence of spiropyran-based sensors, by incorporating an additional fluorophore into the sensor design. We report a 5-membered monoazacrown bearing spiropyran with metal ion specificity, modified to incorporate the pyrene fluorophore. The effect of N-indole pyrene modification on the behavior of the spiropyran molecule is explored, with absorbance and fluorescence emission characterization. This first generation sensor provides an insight into fluorescence-enhancement of spiropyran molecules.

  15. Reverse logistics - a framework

    OpenAIRE

    Brito, Marisa; Dekker, Rommert

    2002-01-01

    textabstractIn this paper we define and compare Reverse Logistics definitions. We start by giving an understanding framework of Reverse Logistics: the why-what-how. By this means, we put in context the driving forces for Reverse Logistics, a typology of return reasons, a classification of products, processes and actors. In addition we provide a decision framework for Reverse Logistics and we present it according to long, medium and short term decisions, i.e. strategic-tactic-operational decis...

  16. Reverse cholesterol transport revisited

    Institute of Scientific and Technical Information of China (English)

    Astrid; E; van; der; Velde

    2010-01-01

    Reverse cholesterol transport was originally described as the high-density lipoprotein-mediated cholesterol flux from the periphery via the hepatobiliary tract to the intestinal lumen, leading to fecal excretion. Since the introduction of reverse cholesterol transport in the 1970s, this pathway has been intensively investigated. In this topic highlight, the classical reverse cholesterol transport concepts are discussed and the subject reverse cholesterol transport is revisited.

  17. Reverse logistics - a framework

    NARCIS (Netherlands)

    M.P. de Brito (Marisa); R. Dekker (Rommert)

    2002-01-01

    textabstractIn this paper we define and compare Reverse Logistics definitions. We start by giving an understanding framework of Reverse Logistics: the why-what-how. By this means, we put in context the driving forces for Reverse Logistics, a typology of return reasons, a classification of product

  18. Fundamentals of fluorescence and fluorescence microscopy.

    Science.gov (United States)

    Wolf, David E

    2013-01-01

    This chapter discusses the fundamental physics of fluorescence. The application of fluorescence to microscopy represents an important transition in the development of microscopy, particularly as it applies to biology. It enables quantitating the amounts of specific molecules within a cell, determining whether molecules are complexing on a molecular level, measuring changes in ionic concentrations within cells and organelles, and measuring molecular dynamics. This chapter also discusses the issues important to quantitative measurement of fluorescence and focuses on four of quantitative measurements of fluorescence--boxcar-gated detection, streak cameras, photon correlation, and phase modulation. Although quantitative measurement presents many pitfalls to the beginner, it also presents significant opportunities to one skilled in the art. This chapter also examines how fluorescence is measured in the steady state and time domain and how fluorescence is applied in the modern epifluorescence microscope.

  19. A NEW REVERSE PHASE HPLC METHOD WITH FLUORESCENT ...

    African Journals Online (AJOL)

    Preferred Customer

    (Tuttlingen, Germany) was utilized to centrifuge plasma samples. ... was dissolved in 100 mL of double distilled water to produce its stock solution (1 .... By this measure, the peak tailing which results from ion-exchange interactions was easily.

  20. Folding and unfolding of a non-fluorescent mutant of green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Wielgus-Kutrowska, Beata [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Narczyk, Marta [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Buszko, Anna [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Bzowska, Agnieszka [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki and Wigury 93, 02-089 (Poland); Clark, Patricia L [Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556-5670 (United States)

    2007-07-18

    Green fluorescent protein (GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and it remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; i.e., the principles of microscopic reversibility do not apply. We have generated the mutant S65T/G67A-GFP, which is unable to efficiently form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of guanidinium hydrochloride (GdnHCl). The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of 1,1'-bis(4-anilino-5-naphthalenesulphonic acid) (bis-ANS). Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements that S65T/G67A-GFP folding is largely reversible. During equilibrium folding, the first step is the formation of a molten globule, prone to aggregation.

  1. Molecular Quantification of Response to Therapy and Remission Status in TEL-AML 1-Positive Childhood ALL by Real-time Reverse Transcription Polymerase Chain R eaction%利用实时反向转录PCR的分子定量监测TEL-AML1+儿童急性淋巴细胞性白血病的治疗反应和缓解状况

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@ 运用分子生物学技术及流式细胞术,对治疗反应和微小残留白血病病灶(MRD)进行灵敏的定量分析,可以更加可靠地预测儿童急性淋巴细胞性白血病(ALL)患者的预后及复发风险 .分子水平的研究大多数利用T细胞受体(TCR)和免疫球蛋白基因重排作为唯一克隆特异性标志来监测MRD,但这种DNA水平上的研究方法时间长、费用高而且工作量大.在儿童ALL 中,有35%~45%出现染色体易位,形成融合基因,出现嵌合体转录本以及表达相应的蛋白质 .儿童初发前B细胞-急性淋巴细胞性白血病(BCP-ALL)最常见的基因重排是隐性易位t(1 2;21)(p13;q22),可导致21号染色体上的AML1序列与12号染色体的TEL基因融合.TEL和AM L1这两种转录因子均为正常造血所必需,而且它们的基因部位分别与急性或慢性白血病及骨髓增生异常综合征相关的染色体易位有关.

  2. Reviews in fluorescence 2010

    CERN Document Server

    Geddes, Chris D

    2011-01-01

    ""Reviews in Fluorescence 2010"", the seventh volume of the book serial from Springer, serves as a comprehensive collection of current trends and emerging hot topics in the field of fluorescence and closely related disciplines. It summarizes the year's progress in fluorescence and its applications, with authoritative analytical reviews specialized enough to be attractive to professional researchers, yet also appealing to the wider audience of scientists in related disciplines of fluorescence. ""Reviews in Fluorescence"" offers an essential reference material for any lab working in the fluoresc

  3. Principles of fluorescence techniques

    CERN Document Server

    2016-01-01

    Fluorescence techniques are being used and applied increasingly in academics and industry. The Principles of Fluorescence Techniques course will outline the basic concepts of fluorescence techniques and the successful utilization of the currently available commercial instrumentation. The course is designed for students who utilize fluorescence techniques and instrumentation and for researchers and industrial scientists who wish to deepen their knowledge of fluorescence applications. Key scientists in the field will deliver theoretical lectures. The lectures will be complemented by the direct utilization of steady-state and lifetime fluorescence instrumentation and confocal microscopy for FLIM and FRET applications provided by leading companies.

  4. Fluorescence correlation spectroscopy

    NARCIS (Netherlands)

    Hink, M.A.; Verveer, P.J.

    2015-01-01

    Fluorescence fluctuation spectroscopy techniques allow the quantification of fluorescent molecules present at the nanomolar concentration level. After a brief introduction to the technique, this chapter presents a protocol including background information in order to measure and quantify the molecul

  5. Reversible cortical blindness: posterior reversible encephalopathy syndrome.

    Science.gov (United States)

    Bandyopadhyay, Sabyasachi; Mondal, Kanchan Kumar; Das, Somnath; Gupta, Anindya; Biswas, Jaya; Bhattacharyya, Subir Kumar; Biswas, Gautam

    2010-11-01

    Cortical blindness is defined as visual failure with preserved pupillary reflexes in structurally intact eyes due to bilateral lesions affecting occipital cortex. Bilateral oedema and infarction of the posterior and middle cerebral arterial territory, trauma, glioma and meningioma of the occipital cortex are the main causes of cortical blindness. Posterior reversible encephalopathy syndrome (PRES) refers to the reversible subtype of cortical blindness and is usually associated with hypertension, diabetes, immunosuppression, puerperium with or without eclampsia. Here, 3 cases of PRES with complete or partial visual recovery following treatment in 6-month follow-up are reported.

  6. Safe biodegradable fluorescent particles

    Science.gov (United States)

    Martin, Sue I.; Fergenson, David P.; Srivastava, Abneesh; Bogan, Michael J.; Riot, Vincent J.; Frank, Matthias

    2010-08-24

    A human-safe fluorescence particle that can be used for fluorescence detection instruments or act as a safe simulant for mimicking the fluorescence properties of microorganisms. The particle comprises a non-biological carrier and natural fluorophores encapsulated in the non-biological carrier. By doping biodegradable-polymer drug delivery microspheres with natural or synthetic fluorophores, the desired fluorescence can be attained or biological organisms can be simulated without the associated risks and logistical difficulties of live microorganisms.

  7. Introduction to reversible computing

    CERN Document Server

    Perumalla, Kalyan S

    2013-01-01

    Few books comprehensively cover the software and programming aspects of reversible computing. Filling this gap, Introduction to Reversible Computing offers an expanded view of the field that includes the traditional energy-motivated hardware viewpoint as well as the emerging application-motivated software approach. Collecting scattered knowledge into one coherent account, the book provides a compendium of both classical and recently developed results on reversible computing. It explores up-and-coming theories, techniques, and tools for the application of rever

  8. Reversible Logic Circuit Synthesis

    CERN Document Server

    Shende, V V; Markov, I L; Prasad, A K; Hayes, John P.; Markov, Igor L.; Prasad, Aditya K.; Shende, Vivek V.

    2002-01-01

    Reversible, or information-lossless, circuits have applications in digital signal processing, communication, computer graphics and cryptography. They are also a fundamental requirement for quantum computation. We investigate the synthesis of reversible circuits that employ a minimum number of gates and contain no redundant input-output line-pairs (temporary storage channels). We propose new constructions for reversible circuits composed of NOT, Controlled-NOT, and TOFFOLI gates (the CNT gate library) based on permutation theory. A new algorithm is given to synthesize optimal reversible circuits using an arbitrary gate library. We also describe much faster heuristic algorithms. We also pursue applications of the proposed techniques to the synthesis of quantum circuits.

  9. 伴有ins(8;21)(q22;q22.1q22.3)髓系白血病的荧光原位杂交研究%Fluorescence in situ hybridization studies on a myeloid leukemia patient with ins (8;21) (q22;q22.1q22.3)

    Institute of Scientific and Technical Information of China (English)

    吴亚芳; 马勤芬; 薛永权; 白淑潇; 张俊; 姚利; 王勇; 仇惠英; 沈娟; 潘金兰

    2009-01-01

    色体亚端粒探针分别进行3种FISH分析;应用逆转录聚合酶链反应(reverse-transcription polymerase chain reaction,RT-PCR)检测AML1-ETO融合转录本.结果 核型分析显示46,XX,ins(8;21)(q22;q22.1q22.3)[73/46,XX[3];FISH分析证实ins(8;21)(q22;q22.1q22.3);RT-PCR分析检出AML1-ETO融合转录本.结论 按照世界卫生组织分型标准,此例应重新诊断为急性髓系白血病,FISH和RT-PCR在证实ins(8;21)中起重要作用.

  10. Reverse Core Engine with Thrust Reverser

    Science.gov (United States)

    Suciu, Gabriel L. (Inventor); Chandler, Jesse M. (Inventor)

    2017-01-01

    An engine system has a gas generator, a bi-fi wall surrounding at least a portion of the gas generator, a casing surrounding a fan, and the casing having first and second thrust reverser doors which in a deployed position abut each other and the bi-fi wall.

  11. Quantum reverse hypercontractivity

    Energy Technology Data Exchange (ETDEWEB)

    Cubitt, Toby [Department of Computer Science, University College London, London, United Kingdom and Centre for Quantum Information and Foundations, DAMTP, University of Cambridge, Cambridge (United Kingdom); Kastoryano, Michael [NBIA, Niels Bohr Institute, University of Copenhagen, 2100 Copenhagen (Denmark); Montanaro, Ashley [School of Mathematics, University of Bristol, Bristol (United Kingdom); Temme, Kristan [Institute for Quantum Information and Matter, California Institute of Technology, Pasadena, California 91125 (United States)

    2015-10-15

    We develop reverse versions of hypercontractive inequalities for quantum channels. By generalizing classical techniques, we prove a reverse hypercontractive inequality for tensor products of qubit depolarizing channels. We apply this to obtain a rapid mixing result for depolarizing noise applied to large subspaces and to prove bounds on a quantum generalization of non-interactive correlation distillation.

  12. Clocked Thrust Reversers

    Science.gov (United States)

    Suciu, Gabriel L. (Inventor); Chandler, Jesse M. (Inventor)

    2017-01-01

    An aircraft includes a fuselage including a propulsion system supported within an aft portion. A thrust reverser is mounted proximate to the propulsion system for directing thrust in a direction to slow the aircraft. The thrust reverser directs thrust at an angle relative to a vertical plane to reduce interference on control surfaces and reduce generation of underbody lift.

  13. Atrioventricular Pacemaker Lead Reversal

    Directory of Open Access Journals (Sweden)

    Mehmet K Aktas, MD

    2007-01-01

    Full Text Available During cardiac surgery temporary epicardial atrial and ventricular leads are placed in case cardiac pacing is required postoperatively. We present the first reported series of patients with reversal of atrioventricular electrodes in the temporary pacemaker without any consequent deleterious hemodynamic effect. We review the electrocardiographic findings and discuss the findings that lead to the discovery of atrioventricular lead reversal.

  14. Reversible cerebral vasoconstriction syndrome

    Directory of Open Access Journals (Sweden)

    Saini Monica

    2009-01-01

    Full Text Available Reversible cerebral vasoconstriction syndromes (RCVS are a group of disorders that have in common an acute presentation with headache, reversible vasoconstriction of cerebral arteries, with or without neurological signs and symptoms. In contrast to primary central nervous system vasculitis, they have a relatively benign course. We describe here a patient who was diagnosed with RCVS.

  15. Towards Reversible Sessions

    Directory of Open Access Journals (Sweden)

    Francesco Tiezzi

    2014-06-01

    Full Text Available In this work, we incorporate reversibility into structured communication-based programming, to allow parties of a session to automatically undo, in a rollback fashion, the effect of previously executed interactions. This permits taking different computation paths along the same session, as well as reverting the whole session and starting a new one. Our aim is to define a theoretical basis for examining the interplay in concurrent systems between reversible computation and session-based interaction. We thus enrich a session-based variant of pi-calculus with memory devices, dedicated to keep track of the computation history of sessions in order to reverse it. We discuss our initial investigation concerning the definition of a session type discipline for the proposed reversible calculus, and its practical advantages for static verification of safe composition in communication-centric distributed software performing reversible computations.

  16. Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

    DEFF Research Database (Denmark)

    Østergaard, H.; Henriksen, A.; Hansen, Flemming G.

    2001-01-01

    To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the i......To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease...

  17. Interfacial adsorption of insulin - Conformational changes and reversibility of adsorption

    NARCIS (Netherlands)

    Mollmann, SH; Jorgensen, L; Bukrinsky, JT; Elofsson, U; Norde, W; Frokjaer, S

    2006-01-01

    The adsorption of human insulin to Teflon particles was studied with respect to conformational changes and the reversibility of adsorption was examined by total internal reflection fluorescence (TIRF). Adsorption isotherms for the adsorption of human insulin indicated high affinity adsorption, even

  18. Interfacial adsorption of insulin. Conformational changes and reversibility of adsorption

    NARCIS (Netherlands)

    Mollmann, S.H.; Bukrinsky, J.T.; Elofsson, U.; Norde, W.; Frokjaer, S.

    2006-01-01

    The adsorption of human insulin to Teflon particles was studied with respect to conformational changes and the reversibility of adsorption was examined by total internal reflection fluorescence (TIRF). Adsorption isotherms for the adsorption of human insulin indicated high affinity adsorption, even

  19. Pyrene-based dual-mode fluorescence switches and logic gates that function in solution and film.

    Science.gov (United States)

    Zhou, Weidong; Li, Yongjun; Li, Yuliang; Liu, Huibiao; Wang, Shu; Li, Cuihong; Yuan, Mingjian; Liu, Xiaofeng; Zhu, Daoben

    2006-07-17

    A dual-mode fluorescence switch controlled by external inputs such as protons and metal ions is described, and each state corresponds to a specific fluorescent emission peak. Based on the reversible changes of the fluorescence emission of the switch responding to different external stimuli, the corresponding integrated logic gates and communication networks have been constructed in solid film or in solution.

  20. An algebra of reversible computation

    OpenAIRE

    2016-01-01

    We design an axiomatization for reversible computation called reversible ACP (RACP). It has four extendible modules, basic reversible processes algebra (BRPA), algebra of reversible communicating processes (ARCP), recursion and abstraction. Just like process algebra ACP in classical computing, RACP can be treated as an axiomatization foundation for reversible computation.

  1. An Algebra of Reversible Computation

    OpenAIRE

    Yong WANG

    2014-01-01

    We design an axiomatization for reversible computation called reversible ACP (RACP). It has four extendible modules, basic reversible processes algebra (BRPA), algebra of reversible communicating processes (ARCP), recursion and abstraction. Just like process algebra ACP in classical computing, RACP can be treated as an axiomatization foundation for reversible computation.

  2. An algebra of reversible computation.

    Science.gov (United States)

    Wang, Yong

    2016-01-01

    We design an axiomatization for reversible computation called reversible ACP (RACP). It has four extendible modules: basic reversible processes algebra, algebra of reversible communicating processes, recursion and abstraction. Just like process algebra ACP in classical computing, RACP can be treated as an axiomatization foundation for reversible computation.

  3. Radiation Thermodynamics with Applications to Lasing and Fluorescent Cooling

    Science.gov (United States)

    2005-04-01

    the flux temperatures of the fluorescence and pump radiation, respectively. The reversible Carnot limit is obtained by choosing the equality sign in...into light and can be interpreted as an optically pumped laser running in reverse . Optical pumping in such devices drives the level populations out of...relaxation cycle . The simplest possible level scheme of such an absorber is depicted in Fig. 2. The material has only three energy levels, where level 1

  4. Reversible Data Hiding Techniques

    Directory of Open Access Journals (Sweden)

    Dhananjay Yadav

    2012-03-01

    Full Text Available Reversible data hiding is a technique that is used to hide data inside an image. The data is hidden in such a way that the exact or original data is not visible. The hidden data can be retrieved as and when required. There are several methods that are used in reversible data hiding techniques like Watermarking, Lossless embedding and encryption. In this paper we present a review of reversible watermarking techniques and show different methods that are used to get reversible data hiding technique with higher embedding capacity and invisible objects. Watermark need not be hidden. Watermarking can be applied to 1. Images, 2. Text, 3. Audio/video, 4. Software.

  5. Reversible flowchart languages and the structured reversible program theorem

    DEFF Research Database (Denmark)

    Yokoyama, Tetsuo; Axelsen, Holger Bock; Glück, Robert

    2008-01-01

    Many irreversible computation models have reversible counterparts, but these are poorly understood at present. We introduce reversible flowcharts with an assertion operator and show that any reversible flowchart can be simulated by a structured reversible flowchart using only three control flow o...... justification for low-level machine code for reversible microprocessors as well as high-level block-structured reversible languages. We give examples for both such languages and illustrate them with a lossless encoder for permutations given by Dijkstra....

  6. Adaptive Pairing Reversible Watermarking.

    Science.gov (United States)

    Dragoi, Ioan-Catalin; Coltuc, Dinu

    2016-05-01

    This letter revisits the pairwise reversible watermarking scheme of Ou et al., 2013. An adaptive pixel pairing that considers only pixels with similar prediction errors is introduced. This adaptive approach provides an increased number of pixel pairs where both pixels are embedded and decreases the number of shifted pixels. The adaptive pairwise reversible watermarking outperforms the state-of-the-art low embedding bit-rate schemes proposed so far.

  7. Single-primer fluorescent sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Ruth, J.L.; Morgan, C.A.; Middendorf, L.R.; Grone, D.L.; Brumbaugh, J.A.

    1987-05-01

    Modified linker arm oligonucleotides complementary to standard M13 priming sites were synthesized, labelled with either one, two, or three fluoresceins, and purified by reverse-phase HPLC. When used as primers in standard dideoxy M13 sequencing with /sup 32/P-dNTPs, normal autoradiographic patterns were obtained. To eliminate the radioactivity, direct on-line fluorescence detection was achieved by the use of a scanning 10 mW Argon laser emitting 488 nm light. Fluorescent bands were detected directly in standard 0.2 or 0.35 mm thick polyacrylamide gels at a distance of 24 cm from the loading wells by a photomultiplier tube filtered at 520 nm. Horizontal and temporal location of each band was displayed by computer as a band in real time, providing visual appearance similar to normal 4-lane autoradiograms. Using a single primer labelled with two fluoresceins, sequences of between 500 and 600 bases have been read in a single loading with better than 98% accuracy; up to 400 bases can be read reproducibly with no errors. More than 50 sequences have been determined by this method. This approach requires only 1-2 ug of cloned template, and produces continuous sequence data at about one band per minute.

  8. Tailoring cyanine dark states for improved optically modulated fluorescence recovery.

    Science.gov (United States)

    Mahoney, Daniel P; Owens, Eric A; Fan, Chaoyang; Hsiang, Jung-Cheng; Henary, Maged M; Dickson, Robert M

    2015-04-02

    Cyanine dyes are well-known for their bright fluorescence and utility in biological imaging. However, cyanines also readily photoisomerize to produce nonemissive dark states. Co-illumination with a secondary, red-shifted light source on-resonance with the longer wavelength absorbing dark state reverses the photoisomerization and returns the cyanine dye to the fluorescent manifold, increasing steady-state fluorescence intensity. Modulation of this secondary light source dynamically alters emission intensity, drastically improving detection sensitivity and facilitating fluorescence signals to be recovered from an otherwise overwhelming background. Red and near-IR emitting cyanine derivatives have been synthesized with varying alkyl chain lengths and halogen substituents to alter dual-laser fluorescence enhancement. Photophysical properties and enhancement with dual laser modulation were coupled with density functional calculations to characterize substituent effects on dark state photophysics, potentially improving detection in high background biological environments.

  9. On thermodynamic and microscopic reversibility

    Energy Technology Data Exchange (ETDEWEB)

    Crooks, Gavin E.

    2011-07-12

    The word 'reversible' has two (apparently) distinct applications in statistical thermodynamics. A thermodynamically reversible process indicates an experimental protocol for which the entropy change is zero, whereas the principle of microscopic reversibility asserts that the probability of any trajectory of a system through phase space equals that of the time reversed trajectory. However, these two terms are actually synonymous: a thermodynamically reversible process is microscopically reversible, and vice versa.

  10. External Quality Assessment for the Detection of Measles Virus by Reverse Transcription-PCR Using Armored RNA.

    Directory of Open Access Journals (Sweden)

    Dong Zhang

    Full Text Available In recent years, nucleic acid tests for detection of measles virus RNA have been widely applied in laboratories belonging to the measles surveillance system of China. An external quality assessment program was established by the National Center for Clinical Laboratories to evaluate the performance of nucleic acid tests for measles virus. The external quality assessment panel, which consisted of 10 specimens, was prepared using armored RNAs, complex of noninfectious MS2 bacteriophage coat proteins encapsulated RNA of measles virus, as measles virus surrogate controls. Conserved sequences amplified from a circulating measles virus strain or from a vaccine strain were encapsulated into these armored RNAs. Forty-one participating laboratories from 15 provinces, municipalities, or autonomous regions that currently conduct molecular detection of measles virus enrolled in the external quality assessment program, including 40 measles surveillance system laboratories and one diagnostic reagent manufacturer. Forty laboratories used commercial reverse transcription-quantitative PCR kits, with only one laboratory applying a conventional PCR method developed in-house. The results indicated that most of the participants (38/41, 92.7% were able to accurately detect the panel with 100% sensitivity and 100% specificity. Although a wide range of commercially available kits for nucleic acid extraction and reverse transcription polymerase chain reaction were used by the participants, only two false-negative results and one false-positive result were generated; these were generated by three separate laboratories. Both false-negative results were obtained with tests performed on specimens with the lowest concentration (1.2 × 104 genomic equivalents/mL. In addition, all 18 participants from Beijing achieved 100% sensitivity and 100% specificity. Overall, we conclude that the majority of the laboratories evaluated have reliable diagnostic capacities for the detection

  11. Kinetic analysis of hydrogen production using anaerobic bacteria in reverse micelles

    Energy Technology Data Exchange (ETDEWEB)

    Zhi, Xiaohua; Yang, Haijun; Yuan, Zhuliang; Shen, Jianquan [Beijing National Laboratory for Molecular Sciences (BNLMS), Laboratory of New Materials, Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun North First Street 2, Beijing 100190 (China)

    2010-04-15

    The micellar formation and entrapment of bacteria cell in reverse micelles were investigated by ultraviolet spectrum (UV), fluorescence spectrum, and scanning electron microscope (SEM). The hydrogen production in reverse micelles was confirmed. The Gompertz equation was employed to evaluate the hydrogen-producing behavior in reverse micellar systems. Different systems including dioctyl sulfosuccinate sodium salt (AOT)-isooctane, sodium dodecyl sulfate (SDS)-benzene and SDS-carbon tetrachloride (CCl{sub 4}) reverse micelles were analysized. The results revealed that the maximum rate of hydrogen production (R{sub m}) was also suitable to formulate the relationship between hydrogen-producing rate and hydrogen productivity in reverse micelles. (author)

  12. Fluorescence antibunching microscopy

    CERN Document Server

    Schwartz, Osip

    2011-01-01

    Breaking the diffraction limit in microscopy by utilizing quantum properties of light has been the goal of intense research in the recent years. We propose a quantum superresolution technique based on non-classical emission statistics of fluorescent markers, routinely used as contrast labels for bio-imaging. The technique can be readily implemented using standard fluorescence microscopy equipment.

  13. Fluorescence of atopic allergens

    NARCIS (Netherlands)

    Berrens, L.

    1967-01-01

    Purified atopic allergens have been found to emit flue fluorescence upon irradiation with ultraviolet light of 365 mμ wavelength. The maximum of fluorescence is in the region 445–490 mμ and the intensity is of the same order of magnitude for different atopic allergens. Synthetic model compounds, inc

  14. Fluorescent Lamp Replacement Study

    Science.gov (United States)

    2017-07-01

    Light-emitting diode, LED, lighting, fluorescent, waste reduction, energy conservation, net zero , mercury 16. SECURITY CLASSIFICATION OF: 17. LIMITATION...Center (ATC) to assess the benefits of converting fluorescent tube lighting to light-emitting diode (LED) technology. The report documents the waste ...1-15 SECTION 2. SUBTESTS 2.1 HAZARDOUS WASTE REDUCTION

  15. LEDs for fluorescence microscopy

    NARCIS (Netherlands)

    Young, I.T.; Garini, Y.; Dietrich, H.R.C.; Van Oel, W.; Liqui Lung, G.

    2004-01-01

    Traditional light sources for fluorescence microscopy have been mercury lamps, xenon lamps, and lasers. These sources have been essential in the development of fluorescence microscopy but each can have serious disadvantages: lack of near monochromaticity, heat generation, cost, lifetime of the light

  16. Reversible chemical reactions for single-color multiplexing microscopy.

    Science.gov (United States)

    Brox, Dominik; Schwering, Michael; Engelhardt, Johann; Herten, Dirk-Peter

    2014-08-04

    Recent developments in biology demand an increasing number of simultaneously imaged structures with standard fluorescence microscopy. However, the number of multiplexed channels is limited for most multiplexing modalities, such as spectral multiplexing or fluorescence-lifetime imaging. We propose extending the number of imaging channels by using chemical reactions, controlling the emissive state of fluorescent dyes. As proof of concept, we reversibly switch a fluorescent copper sensor to enable successive imaging of two different structures in the same spectral channel. We also show that this chemical multiplexing is orthogonal to existing methods. By using two different dyes, we combine chemical with spectral multiplexing for the simultaneous imaging of four different structures with only two spectrally different channels. We characterize and discuss the approach and provide perspectives for extending imaging modalities in stimulated emission depletion microscopy, for which spectral multiplexing is technically demanding.

  17. Reversible Communicating Processes

    Directory of Open Access Journals (Sweden)

    Geoffrey Brown

    2016-02-01

    Full Text Available Reversible distributed programs have the ability to abort unproductive computation paths and backtrack, while unwinding communication that occurred in the aborted paths. While it is natural to assume that reversibility implies full state recovery (as with traditional roll-back recovery protocols, an interesting alternative is to separate backtracking from local state recovery. For example, such a model could be used to create complex transactions out of nested compensable transactions where a programmer-supplied compensation defines the work required to "unwind" a transaction. Reversible distributed computing has received considerable theoretical attention, but little reduction to practice; the few published implementations of languages supporting reversibility depend upon a high degree of central control. The objective of this paper is to demonstrate that a practical reversible distributed language can be efficiently implemented in a fully distributed manner. We discuss such a language, supporting CSP-style synchronous communication, embedded in Scala. While this language provided the motivation for the work described in this paper, our focus is upon the distributed implementation. In particular, we demonstrate that a "high-level" semantic model can be implemented using a simple point-to-point protocol.

  18. Evaluation of the Protective Role of XvPrx2 Gene from Xerophyta ...

    African Journals Online (AJOL)

    Miccah

    Reverse transcription polymerase chain reaction (PCR) revealed the ... important role as light harvesting machinery essential for carbon assimilation. Severe dehydration decreases photosynthetic pigments resulting in increased levels of.

  19. CdTe/ZnS量子点荧光可逆调控研究ctDNA与吡柔比星的相互作用%Reversible control of fluorescence on CdTe/ZnS quantum dots using the interaction between pirarubicin and ctDNA

    Institute of Scientific and Technical Information of China (English)

    闫曙光; 刘绍璞; 何佑秋

    2011-01-01

    Glutathione (GSH)-capped CdTe quantum dots (GSH-CdTe/ZnS QDs) were synthesized in aqueous solution. QD particle sizes'and morphological characteristics were studied by atomic force microscope. The results indicate that a narrow size distribution and good dispersivity were obtained. Fluorescence (FL) of GSH-CdTe/ZnS QDs can be effectively quenched by binding of pirarubicin (THP) on their surfaces, enabling the photoinduced electron transfer (PET) process from the QDs to THP. Subsequent addition of ctDNA restored FL intensity to the QDs as THP dissociated from the surface and embedded into the ctDNA double helix structure, thus preventing PET. Reversible control of FL was realized for anthraquinone anticancer drugs through induced FL quenching and subsequent ctDNA-induced FL restoration for GSH-CdTe/ZnS QDs. The reaction of GSH-CdTe/ZnS QDs-THP-ctDNA is discussed on the basis of FL, resonance Rayleigh scattering and ultraviolet-visible absorption spectra and the spectroscopic method developed was used to investigate the interaction between anthraquinone anticancer drugs and DNA.%水相合成了谷胱甘肽修饰的CdTe/ZnS量子点(GSH-CdTe/ZnS QDs).从原子力显徽镜照片可以看出,合成的核壳型GSH-CdTe/ZnS QDs具有良好的分散性.当吡柔比星与GSH-CdTe/ZnS QDs相互作用时,吡柔比星吸附在GSH-CdTe/ZnS QDs的表面,通过光诱导电子转移的方式猝灭GSH-CdTe/ZnS QDs的荧光.然后向GSH-CdTe/ZnS QDs-吡柔比星体系中加入ctDNA,吡柔比星从GSH-CdTe/ZnS QDs表面脱落后嵌入ctDNA双螺旋结构中,光诱导电子转移过程被阻断,GSH-CdTe/ZnS QDs的荧光恢复.根据GSH-CdTe/ZnSQDs荧光的猝灭和恢复,实现了量子点荧光的可逆调控.结合共振瑞利散射和紫外吸收光谱,讨论了GSH-CdTe/ZnS QDs吡柔比星-ctDNA的相互作用机理,建立了一种研究蒽醌类抗癌药物与核酸相互作用的光谱方法.

  20. 实时荧光RT-PCR检测不同标本中甲型H1N1流感病毒核酸的临床意义%Clinical significance of fluorescent real-time RT-PCR in the detection of influenza A virus (H1N1) nucleic acid in different types of specimens

    Institute of Scientific and Technical Information of China (English)

    陈双峰; 陈海英; 张颖新; 郑丽丽

    2011-01-01

    Objective To explore the relationship of influenza A virus ( H1N1) nucleic acid with rapid diagnosis of influenza A ( H1N1) and the progress of disease course by the detection of viral nucleic acid in different specimens from patients with influenza A ( H1N1). Methods Fluorescent real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect influenza A virus ( H1N1) nucleic acid in throat swabs, plasma and secretion of lower respiratory tract from 118 patients with influenza A ( H1N1) 48 hours after onset and (or) during convalescence respectively. Results Viral nucleic acid was detected in throat swabs from all 118 patients 48 hours after onset,secretion of lower respiratory tract from 23 severe cases and plasma from 15 severe cases. Coriclusion Fluorescent real -time RT-PCR could be used for the rapid detection of influenza A virus ( H1N1) infection. Influenza A virus (H1N1) nucleic acid,when be detected in plasma,may be the indication of the severe cases.%目的 通过检测甲型H1N1流感感染患者不同标本中甲型H1N1流感病毒核酸,了解其对甲型H1N1流感的快速诊断及病程进展的关系.方法 118例甲型H1N1流感患者,采用实时荧光逆转录聚合酶链反应法(RT-PCR)分别检测发病48 h后和(或)恢复期咽拭子、血浆及下呼吸道分泌物中甲型H1N1流感病毒核酸.结果 118例甲型H1N1流感患者发病48 h后咽拭子甲型H1N1流感病毒核酸阳性;23例(23/40)重症患者下呼吸道分泌物中甲型H1N1流感病毒核酸阳性;15例(15/40)重症患者血浆中检测到甲型H1N1流感病毒核酸.结论 实时荧光RT-PCR方法可以快速检测甲型H1N1流感病毒感染,血浆中甲型H1N1流感病毒核酸阳性可能是重症患者的标志.

  1. Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

    Science.gov (United States)

    Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

    2015-06-01

    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

  2. Fluorescence Resonance Energy Transfer Using Spiropyran and Diarylethene Photochromic Acceptors

    Directory of Open Access Journals (Sweden)

    E. A. Jares-Erijman

    2000-03-01

    Full Text Available We describe the preparation and photophysical characterization of two model compounds designed to test a new approach for the quantitative determination of Fluorescence Resonance Energy Transfer (FRET in biological systems. The method enables modulation of FRET by exploiting the unique reversible spectral properties of photochromic diarylethenes and spiropyrans to create switchable energy acceptors.

  3. Radiation controlling reversible window

    Energy Technology Data Exchange (ETDEWEB)

    Gell, H.A. Jr.

    1980-01-01

    A coated glass glazing system is presented including a transparent glass substrate having one surface coated with a radiation absorptive film which is overcoated with a radiation reflective film by a technique which renders the radiation reflective film radiation absorptive at the surface contracting the radiating absorptive film. The coated glass system is used as glazing for storm windows which are adapted to be reversible so that the radiation reflective surface may be exposed to the outside of the dwelling during the warm seasons to prevent excessive solar radiation from entering a dwelling and reversed during cold seasons to absorb solar radiation and utilize it to aid in keeping the dwelling interior warm.

  4. Reversible Photoinhibition in Antarctic Moss during Freezing and Thawing.

    Science.gov (United States)

    Lovelock, C. E.; Jackson, A. E.; Melick, D. R.; Seppelt, R. D.

    1995-01-01

    Tolerance of antarctic moss to freezing and thawing stress was investigated using chlorophyll a fluorescence. Freezing in darkness caused reductions in Fv/Fm (ratio of variable to maximum fluorescence) and Fo (initial fluorescence) that were reversible upon thawing. Reductions in Fv/Fm and Fo during freezing in darkness indicate a reduction in the potential efficiency of photosystem II that may be due to conformational changes in pigment-protein complexes due to desiccation associated with freezing. The absorption of light during freezing further reduced Fv/Fm and Fo but was also reversible. Using dithiothreitol (DTT), which inhibits the formation of the carotenoid zeaxanthin, we found reduced flurorescence quenching during freezing and reduced concentrations of zeaxanthin and antheraxanthin after freezing in DTT-treated moss. Reduced concentrations of zeaxanthin and antheraxanthin in DTT-treated moss were partially associated with reductions in nonphotochemical fluorescence quenching. The reversible photoinhibition observed in antarctic moss during freezing indicates the existence of processes that protect from photoinhibitory damage in environments where freezing temperatures occur in conjunction with high solar radiation levels. These processes may limit the need for repair cycles that require temperatures favorable for enzyme activity. PMID:12228644

  5. Fluorescence and Spectral Imaging

    Directory of Open Access Journals (Sweden)

    Ralph S. DaCosta

    2007-01-01

    Full Text Available Early identification of dysplasia remains a critical goal for diagnostic endoscopy since early discovery directly improves patient survival because it allows endoscopic or surgical intervention with disease localized without lymph node involvement. Clinical studies have successfully used tissue autofluorescence with conventional white light endoscopy and biopsy for detecting adenomatous colonic polyps, differentiating benign hyperplastic from adenomas with acceptable sensitivity and specificity. In Barrett's esophagus, the detection of dysplasia remains problematic because of background inflammation, whereas in the squamous esophagus, autofluorescence imaging appears to be more dependable. Point fluorescence spectroscopy, although playing a crucial role in the pioneering mechanistic development of fluorescence endoscopic imaging, does not seem to have a current function in endoscopy because of its nontargeted sampling and suboptimal sensitivity and specificity. Other point spectroscopic modalities, such as Raman spectroscopy and elastic light scattering, continue to be evaluated in clinical studies, but still suffer the significant disadvantages of being random and nonimaging. A recent addition to the fluorescence endoscopic imaging arsenal is the use of confocal fluorescence endomicroscopy, which provides real-time optical biopsy for the first time. To improve detection of dysplasia in the gastrointestinal tract, a new and exciting development has been the use of exogenous fluorescence contrast probes that specifically target a variety of disease-related cellular biomarkers using conventional fluorescent dyes and novel potent fluorescent nanocrystals (i.e., quantum dots. This is an area of great promise, but still in its infancy, and preclinical studies are currently under way.

  6. Salicyaldehyde-based fluorescent sensors with high sensitivity for amino acids

    Institute of Scientific and Technical Information of China (English)

    Xi Huai Qiang; Zhi Hui Gao; Xue Chuan Wang; Jian Zheng Li; Hui Zong; Chi Min Du

    2011-01-01

    Structurally simple salicylaldehyde-based fluorescent sensors for amino acids have been obtained by one-step or two-step synthesis. These sensors show significant fluorescence enhancement in the presence of many amino acids at concentrations as low as 10 5 mol/L. The reversible reaction of the aldehydes with amino acids to form imines in aqueous solution is proposed to account for the observed fluorescence enhancement.

  7. Sequential Polarity-Reversing Circuit

    Science.gov (United States)

    Labaw, Clayton C.

    1994-01-01

    Proposed circuit reverses polarity of electric power supplied to bidirectional dc motor, reversible electro-mechanical actuator, or other device operating in direction depending on polarity. Circuit reverses polarity each time power turned on, without need for additional polarity-reversing or direction signals and circuitry to process them.

  8. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  9. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  10. Time reversal communication system

    Science.gov (United States)

    Candy, James V.; Meyer, Alan W.

    2008-12-02

    A system of transmitting a signal through a channel medium comprises digitizing the signal, time-reversing the digitized signal, and transmitting the signal through the channel medium. The channel medium may be air, earth, water, tissue, metal, and/or non-metal.

  11. Engineering Encounters: Reverse Engineering

    Science.gov (United States)

    McGowan, Veronica Cassone; Ventura, Marcia; Bell, Philip

    2017-01-01

    This column presents ideas and techniques to enhance your science teaching. This month's issue shares information on how students' everyday experiences can support science learning through engineering design. In this article, the authors outline a reverse-engineering model of instruction and describe one example of how it looked in our fifth-grade…

  12. Reverse Coherent Information

    Science.gov (United States)

    García-Patrón, Raúl; Pirandola, Stefano; Lloyd, Seth; Shapiro, Jeffrey H.

    2009-05-01

    In this Letter we define a family of entanglement distribution protocols assisted by feedback classical communication that gives an operational interpretation to reverse coherent information, i.e., the symmetric counterpart of the well-known coherent information. This leads to the definition of a new entanglement distribution capacity that exceeds the unassisted capacity for some interesting channels.

  13. Reversed extension flow

    DEFF Research Database (Denmark)

    Nielsen, Jens Kromann; Rasmussen, Henrik K.

    2008-01-01

    Afilament stretching rheometer (FSR) was used for measuring the start-up of uni-axial elongational flow followed by reversed bi-axial flow, both with a constant elongational rate. A narrow molecular mass distribution linear polystyrene with a molecular weight of 145 kg / mole wis subjected to the...

  14. REVERSE SUPPLY CHAIN

    Directory of Open Access Journals (Sweden)

    Tomasz DOMAGAŁA

    2013-10-01

    Full Text Available The paper focuses on the presentation of the reverse supply chain, of which the role in the modern business grows along with the increasing number of environmental regulations and possibilities of reducing an operating cost. The paper also describes main problems in developing the profitable chain and possibilities to take an action in order to overcome them.

  15. On reverse hypercontractivity

    CERN Document Server

    Mossel, Elchanan; Sen, Arnab

    2011-01-01

    We study the notion of reverse hypercontractivity. We show that reverse hypercontractive inequalities are implied by standard hypercontractive inequalities as well as by the modified log-Sobolev inequality. Our proof is based on a new comparison lemma for Dirichlet forms and an extension of the Strook-Varapolos inequality. A consequence of our analysis is that {\\em all} simple operators $L=Id-\\E$ as well as their tensors satisfy uniform reverse hypercontractive inequalities. That is, for all $qreverse hypercontractive inequalities established here imply new mixing and isoperimetric results for short random walks in product spaces, for certain card-shufflings, for Glauber dynamics in high-temperat...

  16. Reversing Discrimination: A Perspective

    Science.gov (United States)

    Pati, Gopal; Reilly, Charles W.

    1977-01-01

    Examines the debate over affirmative action and reverse discrimination, and discusses how and why the present dilemma has developed. Suggests that organizations can best address the problem through an honest, in-depth analysis of their organizational structure and management practices. (JG)

  17. Introduction to fluorescence

    CERN Document Server

    Jameson, David M

    2014-01-01

    "An essential contribution to educating scientists in the principles of fluorescence. It will also be an important addition to the libraries of practitioners applying the principles of molecular fluorescence."-Ken Jacobson, Kenan Distinguished Professor of Cell Biology and Physiology, University of North Carolina at Chapel Hill"An exquisite compendium of fluorescence and its applications in biochemistry enriched by a very exciting historical perspective. This book will become a standard text for graduate students and other scientists."-Drs. Zygmunt (Karol) Gryczynski and Ignacy Gryczynski, University of North Texas Health Science Center"… truly a masterwork, combining clarity, precision, and good humor. The reader, novice or expert, will be pleased with the text and will not stop reading. It is a formidable account of the fluorescence field, which has impacted the life sciences so considerably in the last 60 years."-Jerson L. Silva, M.D., Ph.D., Professor and Director, National Institute of Science and Tech...

  18. Functional Fluorescent Organic Nanoparticles

    OpenAIRE

    Campioli, Elisa

    2013-01-01

    This thesis presents an extensive study on fluorescent organic nanoparticles and fluorescent organic binary and ternary nanoassemblies. In particular the attention is focused on the preparation and characterization of organic nanoparticles and new nanocomposites obtained from different types of small organic molecules, their stabilization and the use of these materials for biological and optoelectronics applications. The work deals at the beginning with the description of some methods used...

  19. Reversible quantum cellular automata

    CERN Document Server

    Schumacher, B

    2004-01-01

    We define quantum cellular automata as infinite quantum lattice systems with discrete time dynamics, such that the time step commutes with lattice translations and has strictly finite propagation speed. In contrast to earlier definitions this allows us to give an explicit characterization of all local rules generating such automata. The same local rules also generate the global time step for automata with periodic boundary conditions. Our main structure theorem asserts that any quantum cellular automaton is structurally reversible, i.e., that it can be obtained by applying two blockwise unitary operations in a generalized Margolus partitioning scheme. This implies that, in contrast to the classical case, the inverse of a nearest neighbor quantum cellular automaton is again a nearest neighbor automaton. We present several construction methods for quantum cellular automata, based on unitaries commuting with their translates, on the quantization of (arbitrary) reversible classical cellular automata, on quantum c...

  20. Partial Reversible Gates(PRG) for Reversible BCD Arithmetic

    CERN Document Server

    Thapliyal, Himanshu; Bajpai, Rajnish; Sharma, Kamal K

    2007-01-01

    IEEE 754r is the ongoing revision to the IEEE 754 floating point standard and a major enhancement to the standard is the addition of decimal format. Furthermore, in the recent years reversible logic has emerged as a promising computing paradigm having its applications in low power CMOS, quantum computing, nanotechnology, and optical computing. The major goal in reversible logic is to minimize the number of reversible gates and garbage outputs. Thus, this paper proposes the novel concept of partial reversible gates that will satisfy the reversibility criteria for specific cases in BCD arithmetic. The partial reversible gate is proposed to minimize the number of reversible gates and garbage outputs, while designing the reversible BCD arithmetic circuits.

  1. Reversible hysteresis loop tuning

    Science.gov (United States)

    Berger, A.; Binek, Ch.; Margulies, D. T.; Moser, A.; Fullerton, E. E.

    2006-02-01

    We utilize antiferromagnetically coupled bilayer structures to magnetically tune hysteresis loop properties. Key element of this approach is the non-overlapping switching field distribution of the two magnetic layers that make up the system: a hard magnetic CoPtCrB layer (HL) and a soft magnetic CoCr layer (SL). Both layers are coupled antiferromagnetically through an only 0.6-nm-thick Ru interlayer. The non-overlapping switching field distribution allows the measurement of magnetization reversal in the SL at low fields while keeping the magnetization state of the HL unperturbed. Applying an appropriate high field or high field sequence changes the magnetic state of the HL, which then influences the SL magnetization reversal due to the interlayer coupling. In this way, the position and shape of the SL hysteresis loop can be changed or tuned in a fully reversible and highly effective manner. Here, we study specifically how the SL hysteresis loop characteristics change as we move the HL through an entire high field hysteresis loop sequence.

  2. A Peptoid-Based Fluorescent Sensor for Cyanide Detection

    Directory of Open Access Journals (Sweden)

    Bumhee Lim

    2016-03-01

    Full Text Available Peptoids, N-substituted glycine oligomers, are versatile peptidomimetics with diverse biomedical applications. However, strategies to the development of novel fluorescent peptoids as chemical sensors have not been extensively explored, yet. Here, we synthesized a novel peptoid-based fluorescent probe in which a coumarin moiety was incorporated via copper(I-catalyzed azide-alkyne cycloaddition reaction. Fluorescence of the newly generated coumarin-peptoid was dramatically quenched upon coordination of the Cu2+ ion, and the resulting peptoid-Cu2+ complex exhibited significant Turn-ON fluorescence following the addition of CN−. The rapid and reversible response, combined with cyanide selectivity of the synthesized peptoid, reflects a multistep photo-process and supports its utility as a new type of CN− sensor.

  3. Reversible multi-head finite automata characterize reversible logarithmic space

    DEFF Research Database (Denmark)

    Axelsen, Holger Bock

    2012-01-01

    Deterministic and non-deterministic multi-head finite automata are known to characterize the deterministic and non- deterministic logarithmic space complexity classes, respectively. Recently, Morita introduced reversible multi-head finite automata (RMFAs), and posed the question of whether RMFAs...... characterize reversible logarithmic space as well. Here, we resolve the question affirmatively, by exhibiting a clean RMFA simulation of logarithmic space reversible Turing machines. Indirectly, this also proves that reversible and deterministic multi-head finite automata recognize the same languages....

  4. [Reverse Chaddock sign].

    Science.gov (United States)

    Tashiro, Kunio

    2011-08-01

    It is widely accepted that the Babinski reflex is the most well-known and important pathological reflex in clinical neurology. Among many other pathological reflexes that elicit an upgoing great toe, such as Chaddock, Oppenheim, Gordon, Schaefer, and Stransky, only the Chaddock reflex is said to be as sensitive as the Babinski reflex. The optimal receptive fields of the Babinski and Chaddock reflexes are the lateral plantar surface and the external inframalleolar area of the dorsum, respectively. It has been said that the Babinski reflex, obtained by stroking the sole, is by far the best and most reliable method of eliciting an upgoing great toe. However, the Chaddock reflex, the external malleolar sign, is also considered sensitive and reliable according to the literature and everyday neurological practice. The major problems in eliciting the Babinski reflex by stroking the lateral part of the sole are false positive or negative responses due to foot withdrawal, tonic foot response, or some equivocal movements. On the other hand, according to my clinical experience, the external inframalleolar area, which is the receptive field of the Chaddock reflex, is definitely suitable for eliciting the upgoing great toe. In fact, the newly proposed method to stimulate the dorsum of the foot from the medial to the lateral side, which I term the "reversed Chaddock method," is equally sensitive to demonstrate pyramidal tract involvement. With the "reversed Chaddock method", the receptive field of the Chaddock reflex may be postulated to be in the territory of the sural nerve, which could be supported by the better response obtained on stimulation of the postero-lateral calf than the anterior shin. With regard to the receptive fields of the Babinski and Chaddock reflexes, the first sacral dermatome (S1) is also considered a reflexogenous zone, but since the dermatome shows marked overlapping, the zones vary among individuals. As upgoing toe responses are consistently observed in

  5. Nine New Fluorescent Probes

    Science.gov (United States)

    Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

    1989-06-01

    Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

  6. Stroboscopic fluorescence lifetime imaging.

    Science.gov (United States)

    Holton, Mark D; Silvestre, Oscar R; Errington, Rachel J; Smith, Paul J; Matthews, Daniel R; Rees, Paul; Summers, Huw D

    2009-03-30

    We report a fluorescence lifetime imaging technique that uses the time integrated response to a periodic optical excitation, eliminating the need for time resolution in detection. A Dirac pulse train of variable period is used to probe the frequency response of the total fluorescence per pulse leading to a frequency roll-off that is dependent on the relaxation rate of the fluorophores. The technique is validated by demonstrating wide-field, realtime, lifetime imaging of the endocytosis of inorganic quantum dots by a cancer cell line. Surface charging of the dots in the intra-cellular environment produces a switch in the fluorescence lifetime from approximately 40 ns to technique offers lifetime based imaging at video rates with standard CCD cameras and has application in probing millisecond cell dynamics and in high throughput imaging assays.

  7. Rational design of enhanced photoresistance in a photoswitchable fluorescent protein

    Science.gov (United States)

    Duan, Chenxi; Byrdin, Martin; El Khatib, Mariam; Henry, Xavier; Adam, Virgile; Bourgeois, Dominique

    2015-03-01

    Fluorescent proteins are particularly susceptible to photobleaching, the permanent loss of fluorescence emission resulting from photodestruction of the chromophore. In the case of Reversibly Switchable Fluorescent Proteins (RSFPs), which can be switched back and forth between a non-fluorescent and a fluorescent state, the achievable number of switching cycles is limited by photobleaching, a process known as photofatigue. Photofatigue has become a crucial limitation in a number of advanced applications based on repeated photoswitching of RSFPs, notably in the field of super-resolution fluorescence microscopy. Here, based on our previous structural investigation of photobleaching mechanisms in IrisFP, an RSFP also capable of green-to-red photoconversion, we present the rational design of a single-mutant IrisFP-M159A that displays considerably enhanced photostability. The results suggest that, under moderate illumination intensities, photobleaching of IrisFP-like Anthozoan fluorescent proteins such as EosFP, Dendra or Dronpa derivatives is mainly driven by an oxygen-dependent mechanism resulting in the irreversible sulfoxidation of methionine 159. The photofatigue decay profiles of IrisFP and its photoresistant mutant IrisFP-M159A were investigated in different experimental conditions, in vitro and in cellulo. Although the performance of the mutant was found to be always superior, the results showed switching behaviors strongly dependent on the nanoenvironment. Thus, in general, assessment of photostability and switching properties of RSFPs should be carried out in real experimental conditions.

  8. Reverse Engineering of RFID devices

    OpenAIRE

    Bokslag, Wouter

    2015-01-01

    This paper discusses the relevance and potential impact of both RFID and reverse engineering of RFID technology, followed by a discussion of common protocols and internals of RFID technology. The focus of the paper is on providing an overview of the different approaches to reverse engineering RFID technology and possible countermeasures that could limit the potential of such reverse engineering attempts.

  9. siRNA Downregulates Human Telomerase Reverse Transcriptase Gene and Telomere Length and Telomerase Activity in Human Colon Cancer Colo320 Cells%c-myc靶向siRNA抑制人结直肠癌Colo320细胞的增殖及下调hTERT基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    黄浩; 李秀; 肖宏; 傅雷; 余兰才; 林世和; 易艳东

    2009-01-01

    Objective To study transfected shRNA of c-myc as therapeutic agent in target cell Colo320 which expressed more totomerase activity and to investigate the effect of inhibition on telomerase activity and telomere lengths and tumor cell growth. Methods A plasmid-based polymerase Ⅲ promoter system was used to deliver and express short interfering RNA (siRNA) targeting c-myc in 12o1o320 cells. The c-myc and hTERT mRNA levels were monitored by fluorescence real time reverse transcription-polymerase chain reaction, the protein levels of c-myc and hTERT were examined by Western blot analysis. Mean-while, telomere lengths and telomerase activity were measured by Southern analysis of telomere restric-tion fragment (TRF) length and PCR-ELISA. We also assessed the effects of c-myc silencing on tumor growth by DNA synthesis (3H-thymidine). Results Our data showed that expressions of c-myc and hTERT were decreased in shRNA-transfected cells, and down-regulations of c-myc and hTERT inhibited cell growth, reduced cell telomere lengths, telomerase activity. Conclusion shRNA of c-myc has the a-bility to inhibit telomerase activity, telomere lengths and cell growth with a dose dependent pattern.%目的 探讨发夹状shRNA封阻c-mye基因,抑制人结直肠癌Colo320细胞增殖、生长的状况.方法 针对原癌基因e-myc构建发夹状shRNA的真核表达质粒,并转染人结直肠癌Colo320细胞.荧光定量RT-PCR检测c-myc及细胞端粒酶逆转录酶的mRNA表达,Westemn-blot检测c-myc、hTERT蛋白表达水平.Southern blot检测端粒的长度,PCR-ELISE法检测端粒酶活性.3H-thymidine实验分析DNA合成和细胞增殖.结果 转染细胞增殖、生长皆受到抑制.同时,c-myc和hTERT的mRNA和蛋白表达显著下降,端粒的长度明显缩短,端粒酶活性降低.结论 c-myc的shRNA对人结直肠癌Co1p320细胞的增殖、端粒长度、端粒酶活性有特异性抑制作用,并呈剂量依赖关系.

  10. Nanosecond fluorescence spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Leskovar, B.

    1985-03-01

    This article is a summary of a short course lecture given in conjunction with the 1984 Nuclear Science Symposium. Measuring systems for nanosecond fluorescence spectroscopy using single-photon counting techniques are presented. These involve systems based on relaxation-type spark gap light pulser and synchronously pumped mode-locked dye lasers. Furthermore, typical characteristics and optimization of operating conditions of the critical components responsible for the system time resolution are discussed. A short comparison of the most important deconvolution methods for numerical analysis of experimental data is given particularly with respect to the signal-to-noise ratio of the fluorescence signal. 22 refs., 8 figs.

  11. Reverse Engineering Malicious Applications

    Directory of Open Access Journals (Sweden)

    Ioan Cristian Iacob

    2015-06-01

    Full Text Available Detecting new and unknown malware is a major challenge in today’s software. Security profession. A lot of approaches for the detection of malware using data mining techniques have already been proposed. Majority of the works used static features of malware. However, static detection methods fall short of detecting present day complex malware. Although some researchers proposed dynamic detection methods, the methods did not use all the malware features. In this work, an approach for the detection of new and unknown malware was proposed and implemented. Each sample was reverse engineered for analyzing its effect on the operating environment and to extract the static and behavioral features. 

  12. Reversibly Bistable Flexible Electronics

    KAUST Repository

    Alfaraj, Nasir

    2015-05-01

    Introducing the notion of transformational silicon electronics has paved the way for integrating various applications with silicon-based, modern, high-performance electronic circuits that are mechanically flexible and optically semitransparent. While maintaining large-scale production and prototyping rapidity, this flexible and translucent scheme demonstrates the potential to transform conventionally stiff electronic devices into thin and foldable ones without compromising long-term performance and reliability. In this work, we report on the fabrication and characterization of reversibly bistable flexible electronic switches that utilize flexible n-channel metal-oxide-semiconductor field-effect transistors. The transistors are fabricated initially on rigid (100) silicon substrates before they are peeled off. They can be used to control flexible batches of light-emitting diodes, demonstrating both the relative ease of scaling at minimum cost and maximum reliability and the feasibility of integration. The peeled-off silicon fabric is about 25 µm thick. The fabricated devices are transferred to a reversibly bistable flexible platform through which, for example, a flexible smartphone can be wrapped around a user’s wrist and can also be set back to its original mechanical position. Buckling and cyclic bending of such host platforms brings a completely new dimension to the development of flexible electronics, especially rollable displays.

  13. Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR

    Directory of Open Access Journals (Sweden)

    Delbecchi Louis

    2007-10-01

    Full Text Available Abstract Background In functional genomics, transcript measurement is of fundamental importance. Quantitative reverse transcription polymerase chain reaction (qRT-PCR assays are the most popular technology and depend on the initial molecular step, the reverse transcription (RT. This study provides a complex overview of the influence of elements such as RT systems, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Using qRT-PCR, we compared the efficiency of some commonly used RT systems and measured the production of PCR-amplifiable products and the influence of PCR inhibitor contents. Results The qRT-PCR assays were conducted using the TaqMan system, although we also tested the SYBR Green I chemistry, which is not compatible with all the RT systems. When dealing with low-abundance transcripts, the SuperScript II system generated more detectable molecules than the four other systems tested: Sensiscript, Omniscript, SuperScript III and PowerScript (P P Conclusion This study provides a complex overview of the influence of elements such as RT systems, qRTPCR chemistry, amount of background RNA, and transcript abundance on the efficiency of qRT-PCR. Whereas the most significant influencing factor is the presence of inhibitors in the RT systems, total background RNA is also a major influencing component that affects the PCR reaction. Whenever the aim of a study is to obtain a precise gene expression measurement or to profile the global transcriptome (e.g. microarray, the RT step is critical and should be examined with care.

  14. Reversal Effect and Mechanisms of Recombinant Human Tumor Necrosis Factor-NC Against the Doxorubicin Resistance in Leukemia K562/Doxorubicin Cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Jing-hong; CHEN Bo-hua

    2015-01-01

    Objective: To explore the reversal effect and mechanisms of recombinant human tumor necrosis factor-NC (rhTNF-NC) against the doxorubicin (Dox) resistance in chronic myelogenous leukemia (CML) K562/Dox cells. Methods: The chemo-sensitivity of tumor cells dealt with different concentrations of rhTNF-NC to Dox was detected by tetrazolium dye assay (MTT). The intra-cellular Dox accumulation represented by lfuorescence intensity was determined by lfow cytometry (FCM) at the excitation wave length of 488 nm and emission wave length of 550 nm. The expression of multidrug resistance (MDR)-related genes and proteins was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Results:After being exposed to gradually increasing concentrations of Dox for 10 consecutive months, K562/Dox cells were more resistant to Dox (nearly 132 times) than Dox-sensitive K562 cells. The IC50 of Dox for K562 and K562/Dox cells were (0.04±0.01) and (5.55±0.08) μmol/L, respectively. When K562/Dox cells were treated with rhTNF-NC at 500, 2 500 or 5 000U/mL, the IC50 of Dox was decreased to (2.22±0.34), (1.41±0.13) and (1.04±0.09) μmol/L, respectively. The concentration-response curves were moved upward by the treatment of rhTNF-NC (P Conclusion: rhTNF-NC can effectively augment the drug accumulation in tumor cells. This is due to the up-regulation of TopoIIα and down-regulation of MDR1, MRP and GSTπ at mRNA expression as well as reduction of P-gp and PKCα expression.

  15. Fluorescence Experiments with Quinine

    Science.gov (United States)

    O'Reilly, James E.

    1975-01-01

    Describes a series of experiments which illustrate the analytical capabilities of fluorescence, and outlines two straightforward analyses involving real analyses. These experiments are suitable for an undergraduate instrumental analysis course and require approximately six to seven hours of laboratory time. (MLH)

  16. FLEX: fluorescence explorer

    NARCIS (Netherlands)

    Stoll, M.Ph.; Court, A.J.; Smorenburg, C.; Visser, H.; Crocco, L.; Heilimo, J.; Honig, A.

    1999-01-01

    FLEX is a scientifically driven space mission to provide demonstration/validation of the instrumentation and technique for measuring the natural fluorescence of vegetation in the Fraunhofer lines. The payload consists of high spectral resolution (0.1-0.3 nm) CCD imaging grating spectrometer with two

  17. Fundamentals of reversible flowchart languages

    DEFF Research Database (Denmark)

    Yokoyama, Tetsuo; Axelsen, Holger Bock; Glück, Robert

    2016-01-01

    . Although reversible flowcharts are superficially similar to classical flowcharts, there are crucial differences: atomic steps are limited to locally invertible operations, and join points require an explicit orthogonalizing conditional expression. Despite these constraints, we show that reversible......Abstract This paper presents the fundamentals of reversible flowcharts. They are intended to naturally represent the structure and control flow of reversible (imperative) programming languages in a simple computation model, in the same way classical flowcharts do for conventional languages......, structured reversible flowcharts are as expressive as unstructured ones, as shown by a reversible version of the classic Structured Program Theorem. We illustrate how reversible flowcharts can be concretized with two example programming languages, complete with syntax and semantics: a low-level unstructured...

  18. Fluorescence spectroscopic studies of DNA dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Scalettar, B.A.

    1987-04-01

    Random solvent induced motions of DNA are manifest as nanosecond torsional oscillations of the helix backbone, nanosecond through millisecond bending deformations and overall rotational and translational diffusion of the polymer. Fluorescence spectroscopy is used to study this spectrum of DNA motions while ethidium monoazide was covalently bounded. The steady state fluorescence depolarization data indicate that the covalent monoazide/DNA complex exhibits internal motions characterized by an average angular amplitude of 26 degrees confirming reports of fast torsional oscillations in noncovalent ethidium bromide/DNA systems. Data obtained by use of a new polarized photobleaching recovery technique (FPR) reflect both the rotational dynamics of the polymer and the reversible photochemistry of the dye. To isolate the reorientational motion of the DNA, the FPR experiments were ran in two modes that differ only in the polarization of the bleaching light. A quotient function constructed from the data obtained in these two modes monitors only the rotational component of the FPR recovery. In specific applications those bending deformations of long DNA molecules that have characteristic relaxation times on the order of 100 microseconds have been resolved. A fluorescence correlation technique that relates fluctuations in particle number to center-of-mass motion was used to measure translational diffusion on coefficients of the plasmid PBR322 and a short oligomeric DNA. A theory that describes angular correlation in systems exhibiting cyclic, biologically directed reorientation and random Brownian rotation is developed.

  19. 荧光原位杂交技术可早期有效的预测慢性粒细胞性白血病的预后%Fluorescence in-situ hybridization can provide the early and effective prediction for the prognosis of chronic myeloid leukemia

    Institute of Scientific and Technical Information of China (English)

    彭友帆; 刘洋; 张朝霞

    2015-01-01

    目的:本研究探讨荧光原位杂交技术(FISH)在甲磺酸伊马替尼(IM)治疗慢性粒细胞性白血病(CML)长期预后中的早期预测价值。方法选取60例 IM治疗阶段的 CML 患者骨髓标本,运用骨髓细胞形态学(BM)、FISH、逆转录-聚合酶链反应(RT-PCR)以及常规染色体分析方法(CC)对入组的 CML 患者每3~6个月进行48个月疗效观察。结果运用 FISH 技术在第3个月、第6个月以及第12个月检测融合基因 Bcr-abl 阳性细胞<10%的病例,其在第12个月具有较高的细胞遗传学完全缓解(CCR),同时,在第36个月和第48个月所观察的大多数患者均获得了无事件生存,与 Bcr-abl 阳性细胞百分数>10%的患者组比较,差异具有统计学意义(P <0.05)。相似的,运用 RT-PCR 和 CC 在第3个月和第6个月检测 Bcr-Abl mRNA 以及阳性分裂相百分数,两组病例在第36个月和第48个月的无事件生存率(EFS)差异无统计学意义(P >0.05),仅在第12个月差异具有统计学意义(P <0.05)。结论FISH 分析技术可以为 CML 的长期预后提供早期有效的预测。%Objective To investigate the early predictive significance of fluorescence in-situ hybridization (FISH) in the long-term prognosis of treating chronic myeloid leukemia(CML)by imatinib(IM).Methods A total of 60 patients with CML undergoing IMtreatment were enrolled,their bone marrow specimens were collected,and they were observed per 3-6 months for 48 months for treatment efficiency by bone marrow cell morphology(BM),FISH,reverse transcription polymerase chain reaction (RT-PCR)and karyotyping (CC).Results Applying FISH to detect the patients showed that BCR-ABL positive cell 1 0%,there was statistical significance (P 0.05),and there was statistical significance only in the 1 2th month (P <0.05).Conclusions FISH is able to provide the early and effective prediction for the long-term prognosis

  20. Reversible posterior leukoencephalopathy syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Ja; Yu, Won Jong; Ahn, Kook Jin; Jung, So Lyung; Lee, Yeon Soo; Kim, Ji Chang; Kang, Si Won [The Catholic Univ. of Korea, Taejon (Korea, Republic of); Song, Chang Joon [Chungnam National Univ. School of Medicine, Cheonju (Korea, Republic of); Song, Soon-Young; Koo, Ja Hong [Kwandong Univ. College of Medicine, Myungji Hospital, Seoul (Korea, Republic of); Kim, Man Deuk [College of Medicine Pochon CHA Univ., Seoul (Korea, Republic of)

    2001-10-01

    To review reversible posterior leukoencephalopathy syndrome. We reviewed 22 patients (M:F=3:19; age, 17-46 years) with the characteristic clinical and imaging features of reversible posterior leukoencephalopathy syndrome. All underwent brain MRI, and in three cases both CT and MRI were performed. In one, MRA was obtained, and in eleven, follow-up MR images were obtained. We evaluated the causes of this syndrome, its clinical manifestations, and MR findings including the locations of lesions, the presence or absence of contrast enhancement, and the changes seen at follow-up MRI. Of the 22 patients, 13 had eclampsia (six during pregnancy and seven during puerperium). Four were receiving immunosuppressive therapy (three, cyclosporine ; one, FK 506). Four suffered renal failure and one had complicated migraine. The clinical manifestations included headache (n=12), visual disturbance (n=13), seizure (n=15), focal neurologic sign (n=3), and altered mental status (n=2). Fifteen patients had hypertension and the others normotension. MRI revealed that lesions were bilateral (n=20) or unilateral (n=2). In all patients the lesion was found in the cortical and subcortical areas of the parieto-occipital lobes ; other locations were the basal ganglia (n=9), posterior temporal lobe (n=8), frontal lobe (n=5), cerebellum (n=5), pons (n=2), and thalamus (n=1). All lesions were of high signal intensity on T2-weighted images, and of iso to low intensity on T1-weighted images. One was combined with acute hematoma in the left basal ganglia. In eight of 11 patients who underwent postcontrast T1-weighted MRI, there was no definite enhancement ; in one, enhancement was mild, and in tow, patchy. CT studies showed low attenuation, and MRA revealed mild vasospasm. The symptoms of all patients improved. Follow-up MRI in nine of 11 patients depicted complete resolution of the lesions ; in two, small infarctions remained but the extent of the lesions had decreased. Reversible posterior

  1. Reverse photoacoustic standoff spectroscopy

    Science.gov (United States)

    Van Neste, Charles W [Kingston, TN; Senesac, Lawrence R [Knoxville, TN; Thundat, Thomas G [Knoxville, TN

    2011-04-12

    A system and method are disclosed for generating a reversed photoacoustic spectrum at a greater distance. A source may emit a beam to a target and a detector measures signals generated as a result of the beam being emitted on the target. By emitting a chopped/pulsed light beam to the target, it may be possible to determine the target's optical absorbance by monitoring the intensity of light collected at the detector at different wavelengths. As the wavelength of light is changed, the target may absorb or reject each optical frequency. Rejection may increase the intensity at the sensing element and absorption may decrease the intensity. Accordingly, an identifying spectrum of the target may be made with the intensity variation of the detector as a function of illuminating wavelength.

  2. Is Computation Reversible?

    CERN Document Server

    Parker, M C; Parker, Michael C.; Walker, Stuart D.

    2004-01-01

    Recent investigations into the physical nature of information and fundamental limits to information transmission have revealed questions such as the possibility of superluminal data transfer or not; and whether reversible computation (information processing) is feasible. In some respects these uncertainties stem from the determination of whether information is inherent in points of non-analyticity (discontinuities) or smoother functions. The close relationship between information and entropy is also well known, e.g. Brillouin's concept of negentropy (negative entropy) as a measure for information. Since the leading edge of a step-discontinuity propagates in any dispersive medium at the speed of light in vacuum as a precursor to the main body of the dispersed pulse, we propose in this paper to treat information as being intrinsic to points of non-analyticity (discontinuities). This allows us to construct a theory addressing these dilemmas in a fashion consistent with causality, and the fundamental laws of ther...

  3. Reverse Osmosis Optimization

    Energy Technology Data Exchange (ETDEWEB)

    None

    2013-08-01

    This technology evaluation was prepared by Pacific Northwest National Laboratory on behalf of the U.S. Department of Energy’s Federal Energy Management Program (FEMP). The technology evaluation assesses techniques for optimizing reverse osmosis (RO) systems to increase RO system performance and water efficiency. This evaluation provides a general description of RO systems, the influence of RO systems on water use, and key areas where RO systems can be optimized to reduce water and energy consumption. The evaluation is intended to help facility managers at Federal sites understand the basic concepts of the RO process and system optimization options, enabling them to make informed decisions during the system design process for either new projects or recommissioning of existing equipment. This evaluation is focused on commercial-sized RO systems generally treating more than 80 gallons per hour.

  4. Reverse Osmosis Optimization

    Energy Technology Data Exchange (ETDEWEB)

    McMordie Stoughton, Kate; Duan, Xiaoli; Wendel, Emily M.

    2013-08-26

    This technology evaluation was prepared by Pacific Northwest National Laboratory on behalf of the U.S. Department of Energy’s Federal Energy Management Program (FEMP). ¬The technology evaluation assesses techniques for optimizing reverse osmosis (RO) systems to increase RO system performance and water efficiency. This evaluation provides a general description of RO systems, the influence of RO systems on water use, and key areas where RO systems can be optimized to reduce water and energy consumption. The evaluation is intended to help facility managers at Federal sites understand the basic concepts of the RO process and system optimization options, enabling them to make informed decisions during the system design process for either new projects or recommissioning of existing equipment. This evaluation is focused on commercial-sized RO systems generally treating more than 80 gallons per hour.¬

  5. Multiple stimulus reversible hydrogels

    Science.gov (United States)

    Gutowska, Anna; Krzyminski, Karol J.

    2003-12-09

    A polymeric solution capable of gelling upon exposure to a critical minimum value of a plurality of environmental stimuli is disclosed. The polymeric solution may be an aqueous solution utilized in vivo and capable of having the gelation reversed if at least one of the stimuli fall below, or outside the range of, the critical minimum value. The aqueous polymeric solution can be used either in industrial or pharmaceutical environments. In the medical environment, the aqueous polymeric solution is provided with either a chemical or radioisotopic therapeutic agent for delivery to a specific body part. The primary advantage of the process is that exposure to one environmental stimuli alone will not cause gelation, thereby enabling the therapeutic agent to be conducted through the body for relatively long distances without gelation occurring.

  6. A Reversible Processor Architecture and its Reversible Logic Design

    DEFF Research Database (Denmark)

    Thomsen, Michael Kirkedal; Axelsen, Holger Bock; Glück, Robert

    2012-01-01

    We describe the design of a purely reversible computing architecture, Bob, and its instruction set, BobISA. The special features of the design include a simple, yet expressive, locally-invertible instruction set, and fully reversible control logic and address calculation. We have designed...... an architecture with an ISA that is expressive enough to serve as the target for a compiler from a high-level structured reversible programming language. All-in-all, this paper demonstrates that the design of a complete reversible computing architecture is possible and can serve as the core of a programmable...

  7. Synthesis and Fluorescence Spectra of Triazolylcoumarin Fluorescent Dyes

    Institute of Scientific and Technical Information of China (English)

    PENG Xian-fu; LI Hong-qi

    2009-01-01

    Much attention is devoted to fluorescent dyes especially those with potential in versatile applications. Reactions under "click" conditions between nonfluorescent 3 - azidocoumarins and terminal alkynes produced 3 -(1, 2, 3- triazol- 1 - yl)cournarins, a novel type of fluorescent dyes with intense fluorescence. The structures of the new coumarins were characterized by 1H NMR, MS, and IR spectra. Fluorescence spectra measurement demonstrated excellent fluorescence performance of the triazolylcoumarins and this click reaction is a promising candidate for bioconjugation and bioimaging applications since both azide and alkynes are quite inert to biological systems.

  8. Fluorescence nanoscopy. Methods and applications

    OpenAIRE

    Requejo-Isidro, Jose

    2013-01-01

    Fluorescence nanoscopy refers to the experimental techniques and analytical methods used for fluorescence imaging at a resolution higher than conventional, diffraction-limited, microscopy. This review explains the concepts behind fluorescence nanoscopy and focuses on the latest and promising developments in acquisition techniques, labelling strategies to obtain highly detailed super-resolved images and in the quantitative methods to extract meaningful information from them.

  9. Photophysics of thermally activated delayed fluorescence molecules

    Science.gov (United States)

    Dias, Fernando B.; Penfold, Thomas J.; Monkman, Andrew P.

    2017-03-01

    Thermally activated delayed fluorescence (TADF) has recently emerged as one of the most attractive methods for harvesting triplet states in metal-free organic materials for application in organic light emitting diodes (OLEDs). A large number of TADF molecules have been reported in the literature with the purpose of enhancing the efficiency of OLEDs by converting non-emissive triplet states into emissive singlet states. TADF emitters are able to harvest both singlets and triplet states through fluorescence (prompt and delayed), the latter due to the thermally activated reverse intersystem crossing mechanism that allows up-conversion of low energy triplet states to the emissive singlet level. This allows otherwise pure fluorescent OLEDs to overcome their intrinsic limit of 25% internal quantum efficiency (IQE), which is imposed by the 1:3 singlet-triplet ratio arising from the recombination of charges (electrons and holes). TADF based OLEDS with IQEs close to 100% are now routinely fabricated in the green spectral region. There is also significant progress for blue emitters. However, red emitters still show relatively low efficiencies. Despite the significant progress that has been made in recent years, still significant challenges persist to achieve full understanding of the TADF mechanism and improve the stability of these materials. These questions need to be solved in order to fully implement TADF in OLEDs and expand their application to other areas. To date, TADF has been exploited mainly in the field of OLEDs, but applications in other areas, such as sensing and fluorescence microscopies, are envisaged. In this review, the photophysics of TADF molecules is discussed, summarising current methods to characterise these materials and the current understanding of the TADF mechanism in various molecular systems.

  10. Delayed fluorescence in photosynthesis.

    Science.gov (United States)

    Goltsev, Vasilij; Zaharieva, Ivelina; Chernev, Petko; Strasser, Reto J

    2009-01-01

    Photosynthesis is a very efficient photochemical process. Nevertheless, plants emit some of the absorbed energy as light quanta. This luminescence is emitted, predominantly, by excited chlorophyll a molecules in the light-harvesting antenna, associated with Photosystem II (PS II) reaction centers. The emission that occurs before the utilization of the excitation energy in the primary photochemical reaction is called prompt fluorescence. Light emission can also be observed from repopulated excited chlorophylls as a result of recombination of the charge pairs. In this case, some time-dependent redox reactions occur before the excitation of the chlorophyll. This delays the light emission and provides the name for this phenomenon-delayed fluorescence (DF), or delayed light emission (DLE). The DF intensity is a decreasing polyphasic function of the time after illumination, which reflects the kinetics of electron transport reactions both on the (electron) donor and the (electron) acceptor sides of PS II. Two main experimental approaches are used for DF measurements: (a) recording of the DF decay in the dark after a single turnover flash or after continuous light excitation and (b) recording of the DF intensity during light adaptation of the photosynthesizing samples (induction curves), following a period of darkness. In this paper we review historical data on DF research and recent advances in the understanding of the relation between the delayed fluorescence and specific reactions in PS II. An experimental method for simultaneous recording of the induction transients of prompt and delayed chlorophyll fluorescence and decay curves of DF in the millisecond time domain is discussed.

  11. [Posterior reversible encephalopathy syndrome].

    Science.gov (United States)

    Petrović, Branko; Kostić, Vladimir; Sternić, Nadezda; Kolar, Jovo; Tasić, Nebojsa

    2003-01-01

    Reversible Posterior Leukoencephalopathy Syndrome was introduced into clinical practice in 1996 in order to describe unique syndrome, clinically expressed during hypertensive and uremic encephalopathy, eclampsia and during immunosuppressive therapy [1]. First clinical investigations showed that leucoencephalopathy is major characteristic of the syndrome, but further investigations showed no significant destruction in white cerebral tissue [2, 3, 4]. In majority of cases changes are localise in posterior irrigation area of the brain and in the most severe cases anterior region is also involved. Taking into consideration all above mentioned facts, the suggested term was Posterior Reversible Encephalopathy Syndrome (PRES) for the syndrome clinically expressed by neurological manifestations derived from cortical and subcortical changes localised in posterior regions of cerebral hemispheres, cerebral trunk and cerebellum [5]. Patient, aged 53 years, was re-hospitalized in Cardiovascular Institute "Dediwe" two months after successful aorto-coronary bypass performed in June 2001 due to the chest bone infection. During the treatment of the infection (according to the antibiogram) in September 2001, patient in evening hours developed headache and blurred vision. The recorded blood pressure was 210/120 mmHg so antihypertensive treatment was applied (Nifedipin and Furosemid). After this therapy there was no improvement and intensive headache with fatigue and loss of vision developed. Neurological examination revealed cortical blindness and left hemiparesis. Manitol (20%, 60 ccm every 3 hours) and i.v. Nytroglicerin (high blood pressure). Brain CT revealed oedema of parieto-occipital regions of both hemispheres, more emphasized on the right. (Figure 1a, b, c). There was no sign of focal ischemia even in deeper sections (Figure 1d, e, f). Following three days enormous high blood pressure values were registered. On the fourth day the significant clinical improvement occurred

  12. 49 CFR 230.89 - Reverse gear.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Reverse gear. 230.89 Section 230.89 Transportation... Reversing Gear § 230.89 Reverse gear. (a) General provisions. Reverse gear, reverse levers, and quadrants... quadrant. Proper counterbalance shall be provided for the valve gear. (b) Air-operated power reverse...

  13. Aspiration Level and the Reversal of the Preference Reversal Phenomenon.

    Science.gov (United States)

    1986-09-01

    American Economic Review , 69, 623- 638...Grether, D. M., & Plott, C. R. (1982). Economic theory of choice and the preference reversal phenomenon: Reply. The American Economic Review , 72, 575. Har...34 - . • . ...... ., .. . -. -.,- ... , .. ... - ., . . . . .. . ... . . . . . . . *~~~7 T, W.. 1 d~ I t Y ~ VVW ~ Page 26 1 loomes, G., & Sugden, R. (1983). A rationale for preference reversal. The American Economic Review ,

  14. HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

    Directory of Open Access Journals (Sweden)

    Ennifar Eric

    2008-06-01

    Full Text Available Abstract Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1 is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT, is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC – consisting of viral genomic RNA associated with viral proteins (including RT and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF. We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT.

  15. Design of Reversible Sequential Circuit Using Reversible Logic Synthesis

    Directory of Open Access Journals (Sweden)

    Md. Belayet Ali

    2011-12-01

    Full Text Available Reversible logic is one of the most vital issue at present time and it has different areas for its application,those are low power CMOS, quantum computing, nanotechnology, cryptography, optical computing, DNA computing, digital signal processing (DSP, quantum dot cellular auto meta, communication, computer graphics. It is not possible to realize quantum computing without implementation of reversible logic. The main purposes of designing reversible logic are to decrease quantum cost, depth of the circuits and the number of garbage outputs. In this paper, we have proposed a new reversible gate. And we have designed RS flip flop and D flip flop by using our proposed gate and Peres gate. The proposed designs are better than the existing proposed ones in terms of number of reversible gates and garbage outputs. So, this realization is more efficient and less costly than other realizations.

  16. Design of Reversible Sequential Circuit Using Reversible Logic Synthesis

    Directory of Open Access Journals (Sweden)

    Md. Mosharof Hossin

    2012-01-01

    Full Text Available Reversible logic is one of the most vital issue at present time and it has different areas for its application, those are low power CMOS, quantum computing, nanotechnology, cryptography, optical computing, DNA computing, digital signal processing (DSP, quantum dot cellular automata, communication, computer graphics. It is not possible to realize quantum computing without implementation of reversible logic. The main purposes of designing reversible logic are to decrease quantum cost, depth of the circuits and the number of garbage outputs. In this paper, we have proposed a new reversible gate. And we have designedRS flip flop and D flip flop by using our proposed gate and Peres gate. The proposed designs are better than the existing proposed ones in terms of number of reversible gates and garbage outputs. So, this realization is more efficient and less costly than other realizations.

  17. Time Reversal Violation

    Energy Technology Data Exchange (ETDEWEB)

    Quinn, H; /SLAC

    2009-01-27

    This talk briefly reviews three types of time-asymmetry in physics, which I classify as universal, macroscopic and microscopic. Most of the talk is focused on the latter, namely the violation of T-reversal invariance in particle physics theories. In sum tests of microscopic T-invariance, or observations of its violation, are limited by the fact that, while we can measure many processes, only in very few cases can we construct a matched pair of process and inverse process and observe it with sufficient sensitivity to make a test. In both the cases discussed here we can achieve an observable T violation making use of flavor tagging, and in the second case also using the quantum properties of an antisymmetric coherent state of two B mesons to construct a CP-tag. Both these tagging properties depend only on very general properties of the flavor and/or CP quantum numbers and so provide model independent tests for T-invariance violations. The microscopic laws of physics are very close to T-symmetric. There are small effects that give CP- and T-violating processes in three-generation-probing weak decays. Where a T-violating observable can be constructed we see the relationships between T-violation and CP-violation expected in a CPT conserving theory. These microscopic effects are unrelated to the 'arrow of time' that is defined by increasing entropy, or in the time direction defined by the expansion of our Universe.

  18. Enzymatic reactions in reversed micelles

    NARCIS (Netherlands)

    Hilhorst, M.H.

    1984-01-01

    It has been recognised that enzymes in reversed micelles have potential for application in chemical synthesis. Before these expectations will be realised many problems must be overcome. This thesis deals with some of them.In Chapter 1 the present knowledge about reversed micelles and micellar enzymo

  19. Enzyme recovery using reversed micelles.

    NARCIS (Netherlands)

    Dekker, M.

    1990-01-01

    The objective of this study was to develop a liquid-liquid extraction process for the recovery of extracellular enzymes. The potentials of reaching this goal by using reversed micelles in an organic solvent have been investigated.Reversed micelles are aggregates of surfactant molecules containing an

  20. Timescales and Frequencies of Reversible and Irreversible Adhesion Events of Single Bacterial Cells.

    Science.gov (United States)

    Hoffman, Michelle D; Zucker, Lauren I; Brown, Pamela J B; Kysela, David T; Brun, Yves V; Jacobson, Stephen C

    2015-12-15

    In the environment, most bacteria form surface-attached cell communities called biofilms. The attachment of single cells to surfaces involves an initial reversible stage typically mediated by surface structures such as flagella and pili, followed by a permanent adhesion stage usually mediated by polysaccharide adhesives. Here, we determine the absolute and relative timescales and frequencies of reversible and irreversible adhesion of single cells of the bacterium Caulobacter crescentus to a glass surface in a microfluidic device. We used fluorescence microscopy of C. crescentus expressing green fluorescent protein to track the swimming behavior of individual cells prior to adhesion, monitor the cell at the surface, and determine whether the cell reversibly or irreversibly adhered to the surface. A fluorescently labeled lectin that binds specifically to polar polysaccharides, termed holdfast, discriminated irreversible adhesion events from reversible adhesion events where no holdfast formed. In wild-type cells, the holdfast production time for irreversible adhesion events initiated by surface contact (23 s) was 30-times faster than the holdfast production time that occurs through developmental regulation (13 min). Irreversible adhesion events in wild-type cells (3.3 events/min) are 15-times more frequent than in pilus-minus mutant cells (0.2 events/min), indicating the pili are critical structures in the transition from reversible to irreversible surface-stimulated adhesion. In reversible adhesion events, the dwell time of cells at the surface before departing was the same for wild-type cells (12 s) and pilus-minus mutant cells (13 s), suggesting the pili do not play a significant role in reversible adhesion. Moreover, reversible adhesion events in wild-type cells (6.8 events/min) occur twice as frequently as irreversible adhesion events (3.3 events/min), demonstrating that most cells contact the surface multiple times before transitioning from reversible to

  1. What do reversible programs compute?

    DEFF Research Database (Denmark)

    Axelsen, Holger Bock; Glück, Robert

    2011-01-01

    Reversible computing is the study of computation models that exhibit both forward and backward determinism. Understanding the fundamental properties of such models is not only relevant for reversible programming, but has also been found important in other fields, e.g., bidirectional model...... transformation, program transformations such as inversion, and general static prediction of program properties. Historically, work on reversible computing has focussed on reversible simulations of irreversible computations. Here, we take the viewpoint that the property of reversibility itself should...... are not strictly classically universal, but that they support another notion of universality; we call this RTM-universality. Thus, even though the RTMs are sub-universal in the classical sense, they are powerful enough as to include a self-interpreter. Lifting this to other computation models, we propose r...

  2. Optimization of reversible sequential circuits

    CERN Document Server

    Sayem, Abu Sadat Md

    2010-01-01

    In recent years reversible logic has been considered as an important issue for designing low power digital circuits. It has voluminous applications in the present rising nanotechnology such as DNA computing, Quantum Computing, low power VLSI and quantum dot automata. In this paper we have proposed optimized design of reversible sequential circuits in terms of number of gates, delay and hardware complexity. We have designed the latches with a new reversible gate and reduced the required number of gates, garbage outputs, and delay and hardware complexity. As the number of gates and garbage outputs increase the complexity of reversible circuits, this design will significantly enhance the performance. We have proposed reversible D-latch and JK latch which are better than the existing designs available in literature.

  3. Fluorescent probe based on heteroatom containing styrylcyanine: pH-sensitive properties and bioimaging in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xiaodong [School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640 (China); Gao, Ya; Huang, Zhibing; Chen, Xiaohui; Ke, Zhiyong [School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Zhao, Peiliang; Yan, Yichen [Department of Organic Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China); Liu, Ruiyuan, E-mail: ruiyliu@smu.edu.cn [Department of Organic Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China); Qu, Jinqing, E-mail: cejqqu@scut.edu.cn [School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640 (China)

    2015-07-01

    A novel fluorescent probe based on heteroatom containing styrylcyanine is synthesized. The fluorescence of probe is bright green in basic and neutral media but dark orange in strong acidic environments, which could be reversibly switched. Such behavior enables it to work as a fluorescent pH sensor in the solution state and a chemosensor for detecting acidic and basic volatile organic compounds. Analyses by NMR spectroscopy confirm that the protonation or deprotonation of pyridinyl moiety is responsible for the sensing process. In addition, the fluorescent microscopic images of probe in live cells and zebrafish are achieved successfully, suggesting that the probe has good cell membrane permeability and low cytotoxicity. - Graphical abstract: A novel styrylcyanine-based fluorescent pH probe was designed and synthesized, the fluorescence of which is bright green in basic and neutral media but dark orange in strong acidic environments. Such behavior enables it to work as a fluorescent pH sensor in solution states, and a chemosensor for detecting volatile organic compounds with high acidity and basicity in solid state. In addition, it can be used for fluorescent imaging in living cell and living organism. - Highlights: • Bright green fluorescence was observed in basic and neutral media. • Dark orange fluorescence was found in strong acidic environments. • Volatile organic compounds with high acidity and basicity could be detected. • Bioimaging in living cell and living organism was achieved successfully.

  4. Study on three-dimensional fluorescent spectral characteristics of fluoroquinolones in varying media

    Science.gov (United States)

    Liu, Zhihong; Huang, Zuyun; Cai, Ruxiu

    2000-08-01

    Behaviors of fluoroquinolones in varying media were observed by employing their intrinsic fluorescence. Spectral characteristics in reversed micelles (Aerosol OT/ n-octane) were compared with those in aqueous solution and micelles (sodium dodecyl sulfonate). Those differences in interactions between fluoroquinolones and the various media were clearly illustrated by three-dimensional fluorescent spectra. The influences of other environmental factors on spectral characteristics (pH, SDS concentration, etc.) were also investigated.

  5. Reversed polarity patches at the CMB and geomagnetic field reversal

    Institute of Scientific and Technical Information of China (English)

    XU; Wenyao(徐文耀); WEI; Zigang(魏自刚)

    2002-01-01

    The International Geomagnetic Reference Field models (IGRF) for 1900-2000 are used to calculate the geomagnetic field distribution in the Earth' interior from the ground surface to the core-mantle boundary (CMB) under the assumption of insulated mantle. Four reversed polarity patches, as one of the most important features of the CMB field, are revealed. Two patches with +Z polarity (downward) at the southern African and the southern American regions stand out against the background of -Z polarity (upward) in the southern hemisphere, and two patches of -Z polarity at the North Polar and the northern Pacific regions stand out against the +Z background in the northern hemisphere. During the 1900-2000 period the southern African (SAF) patch has quickly drifted westward at a speed of 0.2-0.3°/a; meanwhile its area has expanded 5 times, and the magnetic flux crossing the area has intensified 30 times. On the other hand, other three patches show little if any change during this 100-year period. Extending upward, each of the reversed polarity patches at the CMB forms a chimney-shaped "reversed polarity column" in the mantle with the bottom at the CMB. The height of the SAF column has grown rapidly from 200km in 1900 to 900km in 2000. If the column grows steadily at the same rate in the future, its top will reach to the ground surface in 600-700 years. And then a reversed polarity patch will be observed at the Earth's surface, which will be an indicator of the beginning of a magnetic field reversal. On the basis of this study, one can describe the process of a geomagnetic polarity reversal, the polarity reversal may be observed firstly in one or several local regions; then the areas of these regions expand, and at the same time, other new reversed polarity regions may appear. Thus several poles may exist during a polarity reversal.

  6. Optimized reversible BCD adder using new reversible logic gates

    CERN Document Server

    Bhagyalakshmi, H R

    2010-01-01

    Reversible logic has received great attention in the recent years due to their ability to reduce the power dissipation which is the main requirement in low power digital design. It has wide applications advanced computing, low power CMOS design, Optical information processing, DNA computing, bio information, quantum computation and nanotechnology. This paper presents an optimized reversible BCD adder using a new reversible gate. A comparative result is presented which shows that the proposed design is more optimized in terms of number of gates, number of garbage outputs and quantum cost than the existing designs.

  7. Reversible modulation of gold nanoclusters photoluminescence based on electrochromic poly(methylene blue).

    Science.gov (United States)

    Zhang, Hui; Zhai, Yanling; Dong, Shaojun

    2014-11-01

    Reversible photoluminescence (PL) switches based on a complex of gold nanoclusters and electrochromic poly(methylene blue) (PMB) were realized. The gold nanoclusters PL of hybrid device can be modulated reversibly under electrochemical stimulation. Such an electrochromic device presents several advantages, such as large fluorescence contrast under reduction and oxidation potentials, good reversibility and excellent long-time stability. This simple protocol is anticipated to offer important hints for other nanoclusters and electrochromic materials in the field of photoelectric devices. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Thermal modulation of the monomer/excimer fluorescence for bispyrene molecules through the gel solution transition of an organogel: A thermo-driven molecular fluorescence switch

    Science.gov (United States)

    Wang, Cheng; Wang, Zhuo; Zhang, Deqing; Zhu, Daoben

    2006-09-01

    Reversible modulation of the monomer/excimer emission was observed for bispyrene molecules 1 and 2 through the solution-gel phase transition (with gelator 3 as the LMWG) and accordingly a thermo-driven molecular fluorescence switch can be established.

  9. Fluorescence-Based Sensors

    Science.gov (United States)

    Orellana, Guillermo

    The natural luminescent phenomena (from the Latin words "lumen" and "essentia", i.e., "made of light") such as northern lights (aurora borealis), marine brightness, glow-worms, shining putrid fish scales, "bluish"- appearing water when contained in certain wooden cups (quinine fluorescence), some stones heated at high temperatures with reducing agents (BaS phosphorescence), or light emitted while crushing sugar (triboluminescence) already fascinated our ancestors. Nowadays we understand that ultraviolet and visible emission of light originates from a competitive deactivation pathway of the lowest electronic excited state of atoms and molecules that produces the so called luminescence (the sub-terms fluorescence and phosphorescence just designate whether the return of the excited to the ground state is an "allowed" or "forbidden" process, namely it is fast or slow, the loosely-defined border between them being a 1-μs-1 rate constant). Actually, luminescence is the only method to generate light in the known Universe regardless it is powered by the nuclear reactions in the stars, the ohmical heating in bulbs, an electric discharge, the absorption of light or a (bio)chemical reaction (chemiluminescence).

  10. The TALE Fluorescence Detectors

    Science.gov (United States)

    Jui, Charles

    2009-05-01

    The TALE fluorescence detectors are designed to extend the threshold for fluorescence observation by TA down to 3x10^16 eV. It will comprise two main components. The first is a set of 24 telescopes working in stereo, with an existing TA FD station at ˜6 km separation. These will cover between 3-31 degrees in elevation and have azimuthal coverage maximizing the stereo aperture in the 10^18-10^19 eV energy range. The second component consists of 15 telescopes equipped with 4m diameter mirrors and covering the sky between 31 and 73 degrees in elevation. The larger mirror size pushes the physics threshold down to 3x10^16 eV, and provides view of the shower maximum for the lower energy events. The Tower detector will cover one quadrant in azimuth and operate in hybrid mode with the TALE infill array to provide redundant composition measurements from both shower maximum information and muon-to-electron ratio.

  11. A fluorescence scanning electron microscope

    Directory of Open Access Journals (Sweden)

    Takaaki Kanemaru

    2010-01-01

    Full Text Available Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM and an electron microscope (EM. In the current study, a scanning electron microscope (SEM (JEOL JXA8600 M was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM. In the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative fluorescence and electron microscopy.

  12. NOVEL REVERSIBLE VARIABLE PRECISION MULTIPLIER USING REVERSIBLE LOGIC GATES

    National Research Council Canada - National Science Library

    M. Saravanan; K. Suresh Manic

    2014-01-01

    .... In this study a reversible logic gate based design of variable precision multiplier is proposed which have the greater efficiency in power consumption and speed since the partial products received...

  13. Supercritical fluid reverse micelle separation

    Science.gov (United States)

    Fulton, John L.; Smith, Richard D.

    1993-01-01

    A method of separating solute material from a polar fluid in a first polar fluid phase is provided. The method comprises combining a polar fluid, a second fluid that is a gas at standard temperature and pressure and has a critical density, and a surfactant. The solute material is dissolved in the polar fluid to define the first polar fluid phase. The combined polar and second fluids, surfactant, and solute material dissolved in the polar fluid is maintained under near critical or supercritical temperature and pressure conditions such that the density of the second fluid exceeds the critical density thereof. In this way, a reverse micelle system defining a reverse micelle solvent is formed which comprises a continuous phase in the second fluid and a plurality of reverse micelles dispersed in the continuous phase. The solute material is dissolved in the polar fluid and is in chemical equilibrium with the reverse micelles. The first polar fluid phase and the continuous phase are immiscible. The reverse micelles each comprise a dynamic aggregate of surfactant molecules surrounding a core of the polar fluid. The reverse micelle solvent has a polar fluid-to-surfactant molar ratio W, which can vary over a range having a maximum ratio W.sub.o that determines the maximum size of the reverse micelles. The maximum ratio W.sub.o of the reverse micelle solvent is then varied, and the solute material from the first polar fluid phase is transported into the reverse micelles in the continuous phase at an extraction efficiency determined by the critical or supercritical conditions.

  14. Supercritical fluid reverse micelle separation

    Science.gov (United States)

    Fulton, J.L.; Smith, R.D.

    1993-11-30

    A method of separating solute material from a polar fluid in a first polar fluid phase is provided. The method comprises combining a polar fluid, a second fluid that is a gas at standard temperature and pressure and has a critical density, and a surfactant. The solute material is dissolved in the polar fluid to define the first polar fluid phase. The combined polar and second fluids, surfactant, and solute material dissolved in the polar fluid is maintained under near critical or supercritical temperature and pressure conditions such that the density of the second fluid exceeds the critical density thereof. In this way, a reverse micelle system defining a reverse micelle solvent is formed which comprises a continuous phase in the second fluid and a plurality of reverse micelles dispersed in the continuous phase. The solute material is dissolved in the polar fluid and is in chemical equilibrium with the reverse micelles. The first polar fluid phase and the continuous phase are immiscible. The reverse micelles each comprise a dynamic aggregate of surfactant molecules surrounding a core of the polar fluid. The reverse micelle solvent has a polar fluid-to-surfactant molar ratio W, which can vary over a range having a maximum ratio W[sub o] that determines the maximum size of the reverse micelles. The maximum ratio W[sub o] of the reverse micelle solvent is then varied, and the solute material from the first polar fluid phase is transported into the reverse micelles in the continuous phase at an extraction efficiency determined by the critical or supercritical conditions. 27 figures.

  15. REVERSAL EFFECTS OF MIFEPRISTONE ON MULTIDRUG RESISTANCE(MDR) IN DRUG-RESISTANT BREAST CANCER CELL LINE MCF7/ADR IN VITRO AND IN VIVO

    Institute of Scientific and Technical Information of China (English)

    李大强; 潘丽华; 邵志敏

    2004-01-01

    Objective: To explore the reversal effect of mifepristone on multidrug resistance (MDR) in drug-resistant human breast cancer cell line MCF7/ADR and its mechanisms. Methods: Expression of MDR1 and MDR-associated protein(MRP) mRNA in MCF7/ADR cells was detected using reverse transcription- polymerase chain reaction(RT-PCR). Western blotting was used to assay the protein levels of P-glycoprotein (P-gp) and MRP. Intracellular rhodamine 123 retention and [3H]vincristine (VCR) accumulation were measured by flow cytometry and liquid scintillation counter, respectively. MTT reduction assay was used to determine the sensitivity of cells to the anticancer agent, adriamycin (ADR). Additionally, a MCF7/ADR cell xenograft model was established to assess the reversal effect of mifeprisone on MDR in MCF7/ADR cells in vivo. Results: Miferpristone dose-dependently down- regulated the expression of MDR1 and MRP mRNA in MCF7/ADR cells, accompanied by a significant decrease in the protein levels of P-gp and MRP. After exposure to 5, 10, and 20 μmol/L mifepristone, MCF7/ADR cells showed a 3.87-, 5.81-, and 7.40-fold increase in the accumulation of intracellular VCR(a known substrate of MRP), and a 2.14-, 4.39-, and 5.53-fold increase in the retention of intracellular rhodamine 123(an indicator of P-gp function), respectively. MTT analysis showed that the sensitivity of MCF7/ADR cells to ADR was enhanced by 7.23-, 13.62-, and 20.96-fold after incubation with mifepristone as above-mentioned doses for 96 h. In vivo, mifepristone effectively restored the chemosensitivity of MCF7/ADR cells to ADR. After 8 weeks of administration with ADR(2 mg·kg-1·d-1) alone or in combination with mifepristone(50 mg·kg-1·d-1), the growth inhibitory rate of xenografted tumors in nude mice was 8.08% and 37.25%, respectively. Conclusion: Mifepristone exerts potent reversal effects on MDR in MCF7/ADR cells in vitro and in vivo through down- regulation of MDR1/P-gp and MRP expression and inhibition of P

  16. NOVEL REVERSIBLE VARIABLE PRECISION MULTIPLIER USING REVERSIBLE LOGIC GATES

    OpenAIRE

    M. Saravanan; K. Suresh Manic

    2014-01-01

    Multipliers play a vital role in digital systems especially in digital processors. There are many algorithms and designs were proposed in the earlier works, but still there is a need and a greater interest in designing a less complex, low power consuming, fastest multipliers. Reversible logic design became the promising technologies gaining greater interest due to less dissipation of heat and low power consumption. In this study a reversible logic gate based design of variable precision multi...

  17. Modulation of a fluorescence switch of nanofiber mats containing photochromic spironaphthoxazine and D-{pi}-A charge transfer dye

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun-Mi [BK21 FTIT Department of Advanced Organic Materials and Textile System Engineering, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Gwon, Seon-Young [Department of Advanced Organic Materials Science and Engineering, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Son, Young-A. [BK21 FTIT Department of Advanced Organic Materials and Textile System Engineering, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Kim, Sung-Hoon, E-mail: shokim@knu.ac.kr [Department of Advanced Organic Materials Science and Engineering, Kyungpook National University, Daegu 702-701 (Korea, Republic of); School of Chemistry Science and Technology, Zhanjiang Normal University, Zhanjiang 524048 (China)

    2012-06-15

    Photoswitchable poly(methyl methacrylate) (PMMA) nanofiber mats containing spironaphthoxazine (SPO)/electron donor-{pi}-acceptor (D-{pi}-A) type fluorescent dye (TCF) were prepared by electrospinning. The photoregulated fluorescence switching behaviors of SPO/TCF blended solution and PMMA nanofiber mats containing SPO/TCF were also studied. Not only SPO/TCF blended solution but also PMMA nanofiber mats containing SPO/TCF showed reversible modulation of fluorescence intensity using alternating irradiation with UV and visible light. Highlights: Black-Right-Pointing-Triangle We have designed PMMA nanofiber mats containing a photochromic SPO and fluorescent TCF. Black-Right-Pointing-Triangle Photoswitchable PMMA nanofiber mats containing SPO/TCF were prepared by electrospinning. Black-Right-Pointing-Triangle Reversible modulation of fluorescence intensity was achieved by irradiation with UV/vis.

  18. Fluorescence of ceramic color standards.

    Science.gov (United States)

    Koo, Annette; Clare, John F; Nield, Kathryn M; Deadman, Andrew; Usadi, Eric

    2010-04-20

    Fluorescence has been found in color standards available for use in calibration and verification of color measuring instruments. The fluorescence is excited at wavelengths below about 600?nm and emitted above 700?nm, within the response range of silicon photodiodes, but at the edge of the response of most photomultipliers and outside the range commonly scanned in commercial colorimeters. The degree of fluorescence on two of a set of 12 glossy ceramic tiles is enough to introduce significant error when those tiles have been calibrated in one mode of measurement and are used in another. We report the nature of the fluorescence and the implications for color measurement.

  19. Maternal Gestational Hypertension-Induced Sensitization of Angiotensin II Hypertension Is Reversed by Renal Denervation or Angiotensin-Converting Enzyme Inhibition in Rat Offspring.

    Science.gov (United States)

    Xue, Baojian; Yin, Haifeng; Guo, Fang; Beltz, Terry G; Thunhorst, Robert L; Johnson, Alan Kim

    2017-04-01

    Numerous findings demonstrate that there is a strong association between maternal health during pregnancy and cardiovascular disease in adult offspring. The purpose of the present study was to test whether maternal gestational hypertension modulates brain renin-angiotensin-aldosterone system (RAAS) and proinflammatory cytokines that sensitizes angiotensin II-elicited hypertensive response in adult offspring. In addition, the role of renal nerves and the RAAS in the sensitization process was investigated. Reverse transcription polymerase chain reaction analyses of structures of the lamina terminalis and paraventricular nucleus indicated upregulation of mRNA expression of several RAAS components and proinflammatory cytokines in 10-week-old male offspring of hypertensive dams. Most of these increases were significantly inhibited by either renal denervation performed at 8 weeks of age or treatment with an angiotensin-converting enzyme inhibitor, captopril, in drinking water starting at weaning. When tested beginning at 10 weeks of age, a pressor dose of angiotensin II resulted in enhanced upregulation of mRNA expression of RAAS components and proinflammatory cytokines in the lamina terminalis and paraventricular nucleus and an augmented pressor response in male offspring of hypertensive dams. The augmented blood pressure change and most of the increases in gene expression in the offspring were abolished by either renal denervation or captopril. The results suggest that maternal hypertension during pregnancy enhances pressor responses to angiotensin II through overactivity of renal nerves and the RAAS in male offspring and that upregulation of the brain RAAS and proinflammatory cytokines in these offspring may contribute to maternal gestational hypertension-induced sensitization of the hypertensive response to angiotensin II. © 2017 American Heart Association, Inc.

  20. Comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-PCR assays.

    Directory of Open Access Journals (Sweden)

    Curi Kim

    Full Text Available BACKGROUND: Many acute respiratory illness surveillance systems collect and test nasopharyngeal (NP and/or oropharyngeal (OP swab specimens, yet there are few studies assessing the relative measures of performance for NP versus OP specimens. METHODS: We collected paired NP and OP swabs separately from pediatric and adult patients with influenza-like illness or severe acute respiratory illness at two respiratory surveillance sites in Kenya. The specimens were tested for eight respiratory viruses by real-time reverse transcription-polymerase chain reaction (qRT-PCR. Positivity for a specific virus was defined as detection of viral nucleic acid in either swab. RESULTS: Of 2,331 paired NP/OP specimens, 1,402 (60.1% were positive for at least one virus, and 393 (16.9% were positive for more than one virus. Overall, OP swabs were significantly more sensitive than NP swabs for adenovirus (72.4% vs. 57.6%, p<0.01 and 2009 pandemic influenza A (H1N1 virus (91.2% vs. 70.4%, p<0.01. NP specimens were more sensitive for influenza B virus (83.3% vs. 61.5%, p = 0.02, parainfluenza virus 2 (85.7%, vs. 39.3%, p<0.01, and parainfluenza virus 3 (83.9% vs. 67.4%, p<0.01. The two methods did not differ significantly for human metapneumovirus, influenza A (H3N2 virus, parainfluenza virus 1, or respiratory syncytial virus. CONCLUSIONS: The sensitivities were variable among the eight viruses tested; neither specimen was consistently more effective than the other. For respiratory disease surveillance programs using qRT-PCR that aim to maximize sensitivity for a large number of viruses, collecting combined NP and OP specimens would be the most effective approach.

  1. A comparative study of microbial diversity and community structure in marine sediments using poly(A tailing and reverse transcription PCR

    Directory of Open Access Journals (Sweden)

    Tatsuhiko eHoshino

    2013-06-01

    Full Text Available To obtain a better understanding of metabolically active microbial communities, we tested a molecular ecological approach using poly(A tailing of environmental 16S rRNA, followed by full-length complementary DNA (cDNA synthesis and sequencing to eliminate potential biases caused by mismatching of PCR primer sequences. The RNA pool tested was extracted from marine sediments of the Yonaguni Knoll IV hydrothermal field in the southern Okinawa Trough. The sequences obtained using the ploy(A tailing method were compared statistically and phylogenetically with those obtained using conventional reverse transcription-polymerase chain reaction (RT-PCR with published domain-specific primers. Both methods indicated that Deltaproteobacteria are predominant in sediment (>85% of the total sequence read. The poly(A tailing method indicated that Desulfobacterales were the predominant deltaproteobacteria, while most of the sequences in libraries constructed using RT-PCR were derived from Desulfuromonadales. This discrepancy may have been due to low coverage of Desulfobacterales by the primers used. A comparison of library diversity indices indicated that the poly(A tailing method retrieves more phylogenetically diverse sequences from the environment. The four archaeal 16S rRNA sequences that were obtained using the poly(A tailing method formed deeply branching lineages that were related to Candidatus Parvarchaeum and the Ancient Archaeal Group. These results clearly demonstrate that poly(A tailing followed by cDNA sequencing is a powerful and less biased molecular ecological approach for the study of metabolically active microbial communities.

  2. Cell tracking using a photoconvertible fluorescent protein.

    Science.gov (United States)

    Hatta, Kohei; Tsujii, Hitomi; Omura, Tomomi

    2006-01-01

    The tracking of cell fate, shape and migration is an essential component in the study of the development of multicellular organisms. Here we report a protocol that uses the protein Kaede, which is fluorescent green after synthesis but can be photoconverted red by violet or UV light. We have used Kaede along with confocal laser scanning microscopy to track labeled cells in a pattern of interest in zebrafish embryos. This technique allows the visualization of cell movements and the tracing of neuronal shapes. We provide illustrative examples of expression by mRNA injection, mosaic expression by DNA injection, and the creation of permanent transgenic fish with the UAS-Gal4 system to visualize morphogenetic processes such as neurulation, placode formation and navigation of early commissural axons in the hindbrain. The procedure can be adapted to other photoconvertible and reversible fluorescent molecules, including KikGR and Dronpa; these molecules can be used in combination with two-photon confocal microscopy to specifically highlight cells buried in tissues. The total time needed to carry out the protocol involving transient expression of Kaede by injection of mRNA or DNA, photoconversion and imaging is 2-8 d.

  3. Molecular structure and reversible photodegradation in anthraquinone dyes

    CERN Document Server

    Dhakal, Prabodh

    2016-01-01

    Reversible photodegradation is a process that has been observed in several dye molecules, but the underlying mechanisms are not still well understood. In this contribution, we characterize a series of anthraquinone dyes to determine how self-healing depends on molecular structure. Past studies have used probing techniques that rely on linear absorption, two-photon fluorescence, and amplified spontaneous emission. Each of these probes provide an indirect measure of the populations of the damaged and undamaged species, requiring calibrations or assumptions to be made that might affect the accuracy of the results. The present studies use fluorescence as a probe, which is shown to directly measure the undamaged population. It is found that certain anthraquinone classes share common structural features that are associated with self healing. Furthermore, the time and temperature dependence of photodegradation and self-healing is found to be consistent with the domain model of self healing.

  4. Giant Amplification of Photoswitching by a Few Photons in Fluorescent Photochromic Organic Nanoparticles.

    Science.gov (United States)

    Su, Jia; Fukaminato, Tuyoshi; Placial, Jean-Pierre; Onodera, Tsunenobu; Suzuki, Ryuju; Oikawa, Hidetoshi; Brosseau, Arnaud; Brisset, François; Pansu, Robert; Nakatani, Keitaro; Métivier, Rémi

    2016-03-07

    Controlling or switching the optical signal from a large collection of molecules with the minimum of photons represents an extremely attractive concept. Promising fundamental and practical applications may be derived from such a photon-saving principle. With this aim in mind, we have prepared fluorescent photochromic organic nanoparticles (NPs), showing bright red emission, complete ON-OFF contrast with full reversibility, and excellent fatigue resistance. Most interestingly, upon successive UV and visible light irradiation, the NPs exhibit a complete fluorescence quenching and recovery at very low photochromic conversion levels (photochromic molecule. This "giant amplification of fluorescence photoswitching" originates from efficient intermolecular energy-transfer processes within the NPs.

  5. A Typology of Reverse Innovation

    DEFF Research Database (Denmark)

    von Zedtwitz, Max; Corsi, Simone; Søberg, Peder Veng

    2015-01-01

    taking place in an emerging country. This analytical framework allows recasting of current research at the intersection between innovation and international business. Of the 10 reverse innovation flows, six are new and have not been covered in the literature to date. The study addresses questions......Reverse innovation commonly refers to an innovation initially launched in a developing country and later introduced to an advanced country. Adopting a linear innovation model with the four sequential phases of concept ideation, product development, primary target market introduction, and subsequent......, the paper then introduces a typology of global innovation with 16 different types of innovation flows between advanced and emerging countries, 10 of which are reverse innovation flows. The latter are further differentiated into weak and strong reverse innovation, depending on the number of innovation phases...

  6. Designing the Reverse Supply Chain

    DEFF Research Database (Denmark)

    Gobbi, Chiara

    2011-01-01

    for the reverse supply chain. Design/methodology/approach – In order to identify the relevance of the Fisher model, the model needs to be recast in terms of PRV, which, in this context, is considered the independent variable in the reverse logistics arena. Products defined as innovative in Fisher's taxonomy...... is associated with first-class recovery options (reconditioning and remarketing). When the recovery option is recycling, time is not relevant, the primary objective is cost reduction (efficiency), the chain is centralized, and actors and phases of the reverse chain are determined by the specificity...... of the recycling process. When the recovery option is reconditioning, time is primarily relevant, tradeoffs between costs and time efficiency are necessary, the chain presents a centralized structure, and the presence of other types of actors and phases influences the structure of the reverse supply chain...

  7. Towards a reversible functional language

    DEFF Research Database (Denmark)

    Yokoyama, Tetsuo; Axelsen, Holger Bock; Glück, Robert

    2012-01-01

    first-match policy for case expressions, we can write overlapping patterns in case branches, as is customary in ordinary functional languages, and also in leaf expressions, unlike existing inverse interpreter methods, which enables concise programs. In patterns, the use of a duplication......We identify concepts of reversibility for a functional language by means of a set of semantic rules with specific properties. These properties include injectivity along with local backward determinism, an important operational property for an efficient reversible language. We define a concise...... reversible first-order functional language in which access to the backward semantics is provided to the programmer by inverse function calls. Reversibility guarantees that in this language a backward run (inverse interpretation) is as fast as the corresponding forward run itself. By adopting a symmetric...

  8. An Overview of Reverse Logistics

    Institute of Scientific and Technical Information of China (English)

    WANG Jia-xiang; HE Xin

    2005-01-01

    Until recently, investment in logistics has focused mainly on the flows from companies to markets. Growing concerns for the environment and conserving resources have created new logistical approaches to more effectively manage the distribution function, and make better use of the resources available to an organization. One such approach is reverse logistics, which uses various methods to give scope for a back-load of finished products, components, waste, reusable packing, etc. from consumer to manufacturer. Back-loads allow manufacturers to reduce costs by using the distribution vehicle's return journey to create income or added value. This basic concept is now being developed to create novel solutions to the problems of reducing pollution, costs and vehicle movements, whilst maintaining high customer service levels. In this paper, the idea of reverse logistics is presented; motivations for it are analyzed, several successful practices are demonstrated and some important truths regarding successful reverse logistics are identified, trend of reverse logistics is provided.

  9. CONCEPTUAL ISSUES REGARDING REVERSE LOGISTICS

    National Research Council Canada - National Science Library

    Ioana Olariu

    2014-01-01

    ... of reverse logistics in companies. Many firms attracted by the value available in the flow, have proactively participated in handling returned products at the end of their usefulness or from other parts of the product life cycle...

  10. Laparoscopic reversal of Hartmann's procedure

    DEFF Research Database (Denmark)

    Svenningsen, Peter Olsen; Bulut, Orhan; Jess, Per

    2010-01-01

    A change in procedure from open to laparoscopic reversal of Hartmann's colostomy was implemented at our department between May 2005 and December 2008. The aim of the study was to investigate if this change was beneficial for the patients.......A change in procedure from open to laparoscopic reversal of Hartmann's colostomy was implemented at our department between May 2005 and December 2008. The aim of the study was to investigate if this change was beneficial for the patients....

  11. Assessing Photosynthesis by Fluorescence Imaging

    Science.gov (United States)

    Saura, Pedro; Quiles, Maria Jose

    2011-01-01

    This practical paper describes a novel fluorescence imaging experiment to study the three processes of photochemistry, fluorescence and thermal energy dissipation, which compete during the dissipation of excitation energy in photosynthesis. The technique represents a non-invasive tool for revealing and understanding the spatial heterogeneity in…

  12. X-ray Fluorescence Sectioning

    CERN Document Server

    Cong, Wenxiang

    2012-01-01

    In this paper, we propose an x-ray fluorescence imaging system for elemental analysis. The key idea is what we call "x-ray fluorescence sectioning". Specifically, a slit collimator in front of an x-ray tube is used to shape x-rays into a fan-beam to illuminate a planar section of an object. Then, relevant elements such as gold nanoparticles on the fan-beam plane are excited to generate x-ray fluorescence signals. One or more 2D spectral detectors are placed to face the fan-beam plane and directly measure x-ray fluorescence data. Detector elements are so collimated that each element only sees a unique area element on the fan-beam plane and records the x-ray fluorescence signal accordingly. The measured 2D x-ray fluorescence data can be refined in reference to the attenuation characteristics of the object and the divergence of the beam for accurate elemental mapping. This x-ray fluorescence sectioning system promises fast fluorescence tomographic imaging without a complex inverse procedure. The design can be ad...

  13. Optical Properties of Fluorescent Dyes

    Institute of Scientific and Technical Information of China (English)

    李戎; 陈东辉

    2001-01-01

    Fluorescent dyes have been widely used these years.Because of the special optical performance, conventional CCM systems seem to be unable to predict the recipes of fabrics dyed with fluorescent dyes. In order to enhance the functions of CCM systems, the optical properties of fluorescent dyes in their absorption region were investigated. It has been found that there was a fixed maximum absorption wavelength for each fluorescent dyes whatever its concentration is. Both absorption region and maximum absorption wavelength of the dyes in solution are the same to those in fabric, and that the absorption is directly proportional to the concentration of the dye. So the optical properties obtained in solutions cna be applied for describing the optics performance of fluorescent dyes in fabrics.

  14. Fluorescence calibration method for single-particle aerosol fluorescence instruments

    Science.gov (United States)

    Shipley Robinson, Ellis; Gao, Ru-Shan; Schwarz, Joshua P.; Fahey, David W.; Perring, Anne E.

    2017-05-01

    Real-time, single-particle fluorescence instruments used to detect atmospheric bioaerosol particles are increasingly common, yet no standard fluorescence calibration method exists for this technique. This gap limits the utility of these instruments as quantitative tools and complicates comparisons between different measurement campaigns. To address this need, we have developed a method to produce size-selected particles with a known mass of fluorophore, which we use to calibrate the fluorescence detection of a Wideband Integrated Bioaerosol Sensor (WIBS-4A). We use mixed tryptophan-ammonium sulfate particles to calibrate one detector (FL1; excitation = 280 nm, emission = 310-400 nm) and pure quinine particles to calibrate the other (FL2; excitation = 280 nm, emission = 420-650 nm). The relationship between fluorescence and mass for the mixed tryptophan-ammonium sulfate particles is linear, while that for the pure quinine particles is nonlinear, likely indicating that not all of the quinine mass contributes to the observed fluorescence. Nonetheless, both materials produce a repeatable response between observed fluorescence and particle mass. This procedure allows users to set the detector gains to achieve a known absolute response, calculate the limits of detection for a given instrument, improve the repeatability of the instrumental setup, and facilitate intercomparisons between different instruments. We recommend calibration of single-particle fluorescence instruments using these methods.

  15. Reversible photo-chem-electrotriggered three-state luminescence switching based on core-shell nanostructures

    Science.gov (United States)

    Zhai, Yanling; Zhu, Zhijun; Zhu, Chengzhou; Zhu, Jinbo; Ren, Jiangtao; Wang, Erkang; Dong, Shaojun

    2013-05-01

    Reversible three-state fluorescence switches triggered by light, electricity and chemical inputs based on ``sponges'' of Pyronin Y-doped silica nanoparticles (PYDS) and polyoxometalate K14[Na(H2O)P5W30O110] (Na-POMs) core-shell nanostructures were realized. Under one or two signal inputs, the system exhibited distinct three-state interconvertible automaton, achieving reversible ``on'' and ``off'' luminescence switches via the related luminescence quenching effect. The features of the system correspond to the equivalent circuitry of an IMPLICATION logic gate performing the Boolean operation by using potential and chemical as inputs. Such a multi-chromic device with novel structure possesses several advantages, such as relative low operation voltage, large reproducibility and reversibility, apparent fluorescence contrast, and long-time stability, which make it a suitable candidate for nonvolatile memory devices. In addition, the current protocol for the hybrid film fabrication can be easily extended from the polyoxometalate and organic dyes to other novel nanostructures matched multifunctional stimulus-responsive species and fluorescence materials in the future.Reversible three-state fluorescence switches triggered by light, electricity and chemical inputs based on ``sponges'' of Pyronin Y-doped silica nanoparticles (PYDS) and polyoxometalate K14[Na(H2O)P5W30O110] (Na-POMs) core-shell nanostructures were realized. Under one or two signal inputs, the system exhibited distinct three-state interconvertible automaton, achieving reversible ``on'' and ``off'' luminescence switches via the related luminescence quenching effect. The features of the system correspond to the equivalent circuitry of an IMPLICATION logic gate performing the Boolean operation by using potential and chemical as inputs. Such a multi-chromic device with novel structure possesses several advantages, such as relative low operation voltage, large reproducibility and reversibility, apparent fluorescence

  16. Single Molecule Spectroscopy of Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    2009-01-01

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

  17. REVERSE LOGISTICS RETAIL LEVEL RETURN

    Directory of Open Access Journals (Sweden)

    Ivona Bajor

    2014-06-01

    Full Text Available Conducting scientific research regarding reverse logistics systems includes certain difficulties. Developed logistics systems are aimed at analysing reverse logistics issues and tend to continuously detect differences and oscillations in the flow of returned products and their characteristics. Developing logistics systems, as Croatian, find reverse logistics issues, regarding product returns, significantly complex and very often these issues are not observed as issues of priority. As distributive flow, reverse logistics systems fundaments should be also based on detailed analysis. Analysis in this flow presents amounts, reasons, process flows and quality of returned items. Because of complex product evaluation on individual level, reverse logistics procedures should be implemented as a methodology individually developed for every supply chain subject. This paper presents a research of retail level returns on the Croatian market, where the analysis implicated that the majority of products in return for this level is directed from final consumers and presents noncurrent inventories of distribution chain. The paper will present conducted research regarding characteristics of returns and routing these products from the retail level.

  18. Cylindrical air flow reversal barrier

    Energy Technology Data Exchange (ETDEWEB)

    Woznica, C.; Rodziewicz, M.

    1988-06-01

    Describes an innovative design introduced in the ZMP mine in Zory for quick reversal of ventilation air flow. Geologic mining conditions at the 705 m deep horizon, where the barrier was built, are described. According to the design used until now, a reversal system consisted of safety barriers, ventilation air locks, a ventilation bridge and stopping needed in case of a fire when air flow direction must be reversed. Nine air locks and an expensive concrete ventilation bridge were needed and the air locks had to be operated at 8 points of the region to effect reversal. The new design consists of a 2-storey cylindrical barrier which also fulfills the function of a ventilation bridge. It can be manually or remotely operated by a mechanical or pneumatic system. Tests showed that the new barrier permits immediate air flow reversal while retaining 60% of the original air, which is important in the case of fire and methane hazards. It permits improved seam panelling and splitting of pillars and brings an economy of about 40 million zlotys in construction cost. Design and operation of the barrier is illustrated and ventilation air circulation is explained. 7 figs.

  19. Are all reversible computations tidy?

    CERN Document Server

    Maroney, O J E

    2004-01-01

    It has long been known that to minimise the heat emitted by a deterministic computer during it's operation it is necessary to make the computation act in a logically reversible manner\\cite{Lan61}. Such logically reversible operations require a number of auxiliary bits to be stored, maintaining a history of the computation, and which allows the initial state to be reconstructed by running the computation in reverse. These auxiliary bits are wasteful of resources and may require a dissipation of energy for them to be reused. A simple procedure due to Bennett\\cite{Ben73} allows these auxiliary bits to be "tidied", without dissipating energy, on a classical computer. All reversible classical computations can be made tidy in this way. However, this procedure depends upon a classical operation ("cloning") that cannot be generalised to quantum computers\\cite{WZ82}. Quantum computations must be logically reversible, and therefore produce auxiliary qbits during their operation. We show that there are classes of quantu...

  20. Low Cost Reversible Signed Comparator

    Directory of Open Access Journals (Sweden)

    Farah Sharmin

    2013-10-01

    Full Text Available Nowadays exponential advancement in reversible comp utation has lead to better fabrication and integration process. It has become very popular ove r the last few years since reversible logic circuit s dramatically reduce energy loss. It consumes less p ower by recovering bit loss from its unique input-o utput mapping. This paper presents two new gates called RC-I and RC-II to design an n-bit signed binary comparator where simulation results show that the p roposed circuit works correctly and gives significa ntly better performance than the existing counterparts. An algorithm has been presented in this paper for constructing an optimized reversible n-bit signed c omparator circuit. Moreover some lower bounds have been proposed on the quantum cost, the numbers of g ates used and the number of garbage outputs generated for designing a low cost reversible sign ed comparator. The comparative study shows that the proposed design exhibits superior performance consi dering all the efficiency parameters of reversible logic design which includes number of gates used, quantum cost, garbage output and constant inputs. This proposed design has certainly outperformed all the other existing approaches.

  1. Vasectomy reversal: a clinical update

    Directory of Open Access Journals (Sweden)

    Abhishek P Patel

    2016-01-01

    Full Text Available Vasectomy is a safe and effective method of contraception used by 42-60 million men worldwide. Approximately 3%-6% of men opt for a vasectomy reversal due to the death of a child or divorce and remarriage, change in financial situation, desire for more children within the same marriage, or to alleviate the dreaded postvasectomy pain syndrome. Unlike vasectomy, vasectomy reversal is a much more technically challenging procedure that is performed only by a minority of urologists and places a larger financial strain on the patient since it is usually not covered by insurance. Interest in this procedure has increased since the operating microscope became available in the 1970s, which consequently led to improved patency and pregnancy rates following the procedure. In this clinical update, we discuss patient evaluation, variables that may influence reversal success rates, factors to consider in choosing to perform vasovasostomy versus vasoepididymostomy, and the usefulness of vasectomy reversal to alleviate postvasectomy pain syndrome. We also review the use of robotics for vasectomy reversal and other novel techniques and instrumentation that have emerged in recent years to aid in the success of this surgery.

  2. Cloning and Expression Analysis of Mlo Gene from Pericallis hybrida B. Nord

    Institute of Scientific and Technical Information of China (English)

    Wang Jin-gang; Li Wei; Ren Hong-wei; Lv Yuan-da; Bai Ding; Xu Jing

    2014-01-01

    The full-length Mlo gene was obtained by reverse transcription polymerase chain reaction (RT-PCR) and RACE. The result of sequence analysis indicated that Mlo gene from Pericallis hybrida B. Nord. contained about 1 296 bp open reading frame and encoded 431 amino acids. According to the comparison of the exogenous gene sequences by BLAST analysis and phylogenetic analysis, Mlo gene shared over 85%nucleotide homology and 98%amino acid homology. Finally, through semi-quantitative-PCR and fluorescence quantitative analysis, we found that Mlo gene showed the highest expression levels in leaves and the lowest in roots after inoculated with powdery mildew pathogen for different days.

  3. Dysregulation of CD47 and the ligands thrombospondin 1 and 2 in multiple myeloma

    DEFF Research Database (Denmark)

    Rendtlew Danielsen, Jannie M; Knudsen, Lene Meldgaard; Dahl, Inger Marie

    2007-01-01

    CD47 and thrombospondin 1 and 2 (TSP1 and TSP2) expression were analysed by real-time reverse transcription polymerase chain reaction in fluorescence-activated cell sorted plasma cells (PCs) from patients at consecutive stages of multiple myeloma (MM) development. 80% of MM patients, but only 39......% of patients with monoclonal gammopathy of undetermined significance (MGUS) expressed CD47; median expression level increased 10-fold with progression from MGUS to MM. Elevated TSP1/TSP2 levels occurred in bone marrow cultures from MM patients compared with healthy donors. CD47 and TSP1/TSP2 may have...

  4. FLEX: fluorescence explorer

    Science.gov (United States)

    Stoll, Marc-Ph.; Court, Andrew; Smorenburg, Kees; Visser, Huib; Crocco, Luiggi; Heilimo, Jyro; Honig, Andre

    1999-12-01

    FLEX is a scientifically driven space mission to provide demonstration/validation of the instrumentation and technique for measuring the natural fluorescence of vegetation in the Fraunhofer lines. The payload consists of high spectral resolution (0.1 - 0.3 nm) CCD imaging grating spectrometer with two channels: one in the red (648 - 664 nm) and one in the blue (391 - 438 nm) for working with several Fraunhofer lines. The across track FOV is 8.4 degrees; ground spatial resolution is better than 0.5 X 0.5 km2. To increase the S/N ratio a steering mirror will be used, if necessary, to 'freeze' the image and also to provide plus or minus 4 degrees across track depointing. Calibration is made by viewing the sun via a diffuser plate switched into the telescope field of view. A separate CCD camera will allow cloud detection and scene identification. A TIR radiometer will provide simultaneous surface temperature measurements. The spacecraft, overall mass estimated at 200 kg, is derived from the ASI-MITA bus which provides all the necessary subsystems and stabilized platform. By use of on-board storage, ground requirements for satellite control and data link are minimized; the possibility of local stations for real time reception/distribution is also envisaged. Provisional orbit characteristics are: LEO sun synchronous, 500 - 900 km altitude. Priority will be given to highest revisit frequency on a sufficient number of selected test sites.

  5. Fluorescent blood cell angiography

    Science.gov (United States)

    Ben-nun, Joshua; Constable, Ian J.

    1994-06-01

    Fluorescein angiography is currently the main method for evaluation of the retinal vascular patency. Ashton noted that capillary patency to the small fluorescein molecule may differ from that of the larger red blood cells. He concluded that fluorescein angiography is not able to demonstrate a developing stenosis, that might be the precipitating cause of a later capillary closure in various microvasculopathies. Sarelius et al have shown, in hamster cheek pouch and cremaster muscle, that fluorescently labeled erythrocytes in known concentrations can be used for the direct measurement of capillary flow parameters. The only assumption that this method relies on, is that the labeled cells are rheologically normal and therefore reflect the behavior of the total cell population. We have developed a new method for an in-vivo, real-time demonstration of the blood cell flow in the retinal capillary net. Based on the assumption presented by Sarelius et al, measurement and analysis of the retinal capillary blood cell flow is also possible from the results achieved by the new method.

  6. Fluorescence endoscopy and photodynamic therapy.

    Science.gov (United States)

    Messmann, H; Endlicher, E; Gelbmann, C M; Schölmerich, J

    2002-10-01

    Fluorescence endoscopy is a new technique which allows a better detection of non-visible malignant or premalignant lesions or, those which are difficult to detect. Exogenously applied sensitisers accumulate selectively in malignant lesions and induce fluorescence after illumination with light of adequate wavelength. However, also endogenous fluorophores, different located in malignant or benign lesions, induce a different autofluorescence in these lesions. Tissue fluorescence can be detected by optical sampling of the mucosa using fluorescence spectroscopy or by generating real time fluorescence images with specialised camera systems. Compared to point fluorescence spectroscopy the latter technique enables the screening of large surface areas of mucosa. Meanwhile, fluorescence endoscopy is a widely used technique in urology employing 5-aminolaevulinic acid sensitisation. In gastroenterology, this technique seems promising for the detection of early cancers or dysplasia in patients with Barrett's oesophagus or ulcerative colitis. Using different sensitisers, photodynamic therapy seems to be a promising option for patients with advanced oesophageal cancer and in the palliative treatment of non-resectable bile duct cancer, furthermore for patients with early gastric cancer and dysplasia in Barrett's oesophagus. Probably, by laser light fractionation or a combination of different sensitisers, an enhanced effect can be expected.

  7. 柱前衍生反相高效液相色谱-荧光检测法测定大鼠血浆中的奈替米星%Determination of netilmicin in rat plasma by reversed-phase high performance liquid chromatography with fluorescence detection and pre-column derivatization

    Institute of Scientific and Technical Information of China (English)

    常晓娟; 彭敬东; 刘绍璞; 刘丽敏; 代永矿

    2009-01-01

    建立了一种简便、灵敏的氯甲酸芴甲酯(FMOC-Cl)柱前衍生反相高效液相色谱-荧光检测血浆中奈替米星的新方法,同时研究了其药代动力学.对色谱条件进行了优化,采用ZORBAX Eclipse XDB-C_8柱(150 mm×4.6 mm,5 μm),流动相为乙腈-水(体积比为85∶15),流速为1.0 mL/min,荧光检测激发波长为265 nm,发射波长为315 nm,得到奈替米星的平均加标回收率为96.62% ~100.84%(n=3),对奈替米星检测的线性范围为0.045~8.88 mg/L,相关系数为 0.999 3,方法的日内与日间精密度分别低于3%与3.5%,最低检出限(S/N=3)与定量限(以3倍检出限计)分别为0.01和0.03 mg/L.方法简便、快速、灵敏,样品用量少(30 μL奈替米星血浆溶液已能满足该药含量的测定以及药物代谢的研究),为大鼠体内奈替米星的药代动力学研究提供了可靠的分析手段.%A new, simple and sensitive method based on pre-column derivatization by reversed-phase high performance liquid chromatography (HPLC) is described for the separation and quantification of netilmicin in plasma, using 9-fluorenylmethyl chloroformate (FMOC-Cl) as the derivatization reagent. Its pharacokinetics is also presented. The derivatization modes and chromatographic conditions were optimized. The separation was performed on an Agilent ZOR-BAX Eclipse XDB-C_8 column (150 mm ×4. 6 mm, 5 μm) with a mixture of water-acetonitile (15:85, v/v) as mobile phase and the flow rate was 1.0 mL/min. The excitation wavelength was 265 nm and the emission wavelength was 315 nm. The linear range was 0. 045-8. 88 mg/L and the correlation coefficient (r) was 0. 999 3. The limit of detection (LOD) (S/N = 3) was about 0.01 mg/L, and the limit of quantification was 0.03 mg/L (3LOD) for netilmicin. The relative standard deviation was less than 3% for intra-day assay (n =5) and 3. 5% for inter-day assay (n = 5) and the relative recovery was in the range of 96. 62% - 100. 84% (n = 3). The plasma volume of 30

  8. Laparoscopic reversal of Hartmann's procedure

    DEFF Research Database (Denmark)

    Svenningsen, Peter Olsen; Bulut, Orhan; Jess, Per

    2010-01-01

    INTRODUCTION: A change in procedure from open to laparoscopic reversal of Hartmann's colostomy was implemented at our department between May 2005 and December 2008. The aim of the study was to investigate if this change was beneficial for the patients. MATERIAL AND METHODS: The medical records...... of all patients who underwent reversal of a colostomy after a primary Hartmann's procedure during the period May 2005 to December 2008 were reviewed retrospectively in a case-control study. RESULTS: A total of 43 patients were included. Twenty-one had a laparoscopic and 22 an open procedure. The two...

  9. Reversible Switching of Cooperating Replicators

    Science.gov (United States)

    Urtel, Georg C.; Rind, Thomas; Braun, Dieter

    2017-02-01

    How can molecules with short lifetimes preserve their information over millions of years? For evolution to occur, information-carrying molecules have to replicate before they degrade. Our experiments reveal a robust, reversible cooperation mechanism in oligonucleotide replication. Two inherently slow replicating hairpin molecules can transfer their information to fast crossbreed replicators that outgrow the hairpins. The reverse is also possible. When one replication initiation site is missing, single hairpins reemerge from the crossbreed. With this mechanism, interacting replicators can switch between the hairpin and crossbreed mode, revealing a flexible adaptation to different boundary conditions.

  10. Marburg Virus Reverse Genetics Systems.

    Science.gov (United States)

    Schmidt, Kristina Maria; Mühlberger, Elke

    2016-06-22

    The highly pathogenic Marburg virus (MARV) is a member of the Filoviridae family and belongs to the group of nonsegmented negative-strand RNA viruses. Reverse genetics systems established for MARV have been used to study various aspects of the viral replication cycle, analyze host responses, image viral infection, and screen for antivirals. This article provides an overview of the currently established MARV reverse genetic systems based on minigenomes, infectious virus-like particles and full-length clones, and the research that has been conducted using these systems.

  11. Marburg Virus Reverse Genetics Systems

    Directory of Open Access Journals (Sweden)

    Kristina Maria Schmidt

    2016-06-01

    Full Text Available The highly pathogenic Marburg virus (MARV is a member of the Filoviridae family and belongs to the group of nonsegmented negative-strand RNA viruses. Reverse genetics systems established for MARV have been used to study various aspects of the viral replication cycle, analyze host responses, image viral infection, and screen for antivirals. This article provides an overview of the currently established MARV reverse genetic systems based on minigenomes, infectious virus-like particles and full-length clones, and the research that has been conducted using these systems.

  12. Nickel-hydrogen cell reversal characteristics

    Science.gov (United States)

    Lurie, Charles

    1994-01-01

    Nickel-hydrogen cell reversal characteristics are being studied as part of a TRW program directed towards development of a high current battery cell bypass switch. The following are discussed: cell bypass switch; nickel-hydrogen cell reversal characteristics; and nickel-hydrogen cell chemistry: discharge/reversal and overdischarge (reversal) with nickel and hydrogen precharge.

  13. Murine leukemia virus (MLV replication monitored with fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Bittner Alexandra

    2004-12-01

    Full Text Available Abstract Background Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results We inserted the coding sequences for green fluorescent protein (GFP into the proline-rich region (PRR of the ecotropic envelope protein (Env and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

  14. Fluorescence lifetime measurements of boronate derivatives to determine glucose concentration

    Energy Technology Data Exchange (ETDEWEB)

    Gable, J H

    2000-06-01

    A novel investigation into the fluorescence lifetimes of molecules, both established and newly designed, was performed. These molecules are the basis of a continuous, minimally invasive, glucose sensor based on fluorescence lifetime measurements. This sensor, if coupled with an automated insulin delivery device, would effectively create an artificial pancreas allowing for the constant monitoring and control of glucose levels in a person with diabetes. The proposed sensor includes a fluorescent molecule that changes its' fluorescence properties upon binding selectively and reversibly to glucose. One possible sensor molecule is N-methyl-N-(9-methylene anthryl)-2-methylenephenylboronic acid (AB). The fluorescence intensity of AB was shown to change in response to changing glucose concentrations. (James, 1994) James proposed that when glucose binds to AB the fluorescence intensity increases due to an enhancement of the N{yields}B dative bond which prevents photoinduced electron transfer (PET). PET from the amine (N) to the fluorophore (anthracene) quenches the fluorescence. The dative bond between the boron and the amine can prevent PET by involving the lone pair of electrons on the amine in interactions with the boron rather than allowing them to be transferred to the fluorophore. Results of this research show the average fluorescence lifetime of AB also changes with glucose concentration. It is proposed that fluorescence is due to two components: (1) AB with an enhanced N{yields}B interaction, and no PET, and (2) AB with a weak N{yields}B interaction, resulting in fluorescence quenching by PET. Lifetime measurements of AB as a function of both the pH of the solvent and glucose concentration in the solution were made to characterize this two component system and investigate the nature of the N{yields}B bond. Measurements of molecules similar to AB were also performed in order to isolate behavior of specific AB constituents. These molecules are 9

  15. Modular generation of fluorescent phycobiliproteins.

    Science.gov (United States)

    Wu, Xian-Jun; Chang, Kun; Luo, Juan; Zhou, Ming; Scheer, Hugo; Zhao, Kai-Hong

    2013-06-01

    Phycobiliproteins are brightly-fluorescent light-harvesting pigments for photosynthesis in cyanobacteria and red algae. They are also of interest as fluorescent biomarkers, but their heterologous generation in vivo has previously required multiple transformations. We report here a modular approach that requires only two DNA segments. The first codes for the apo-protein. The second codes for fusions capable of chromophore biosynthesis and its covalent attachment to the apo-protein; it contains the genes of heme oxygenase, a bilin reductase, and a chromophore lyase. Phycobiliproteins containing phycoerythrobilin (λ(fluor) ~ 560 nm), phycourobilin (λ(fluor) ~ 500 nm), phycocyanobilin (λ(fluor) ~ 630 nm) or phycoviolobilin (λ(fluor) ~ 580 nm) were obtained in high yield in E. coli. This approach facilitates chromophorylation studies of phycobiliproteins, as well as their use for fluorescence labeling based on their high fluorescence.

  16. Fluorescence diagnosis in tissue injury

    Science.gov (United States)

    Maciel, Vitória H.; Ferreira, Juliana; Bagnato, Vanderlei S.

    2009-06-01

    Background and Objectives: The paper aim was to evaluate the efficacy of the fluorescence spectroscopy in the detection of UV-induced skin change of Wistar rats. Study Design/ Materials and Methods: In a group male Wistar rats, the skin damage was produced by an UV-C lamp, periodically monitored using the laser-induced fluorescence, until complete healing process. After determining a characteristic emission band present in the fluorescence spectra of the induced injuries, the amplitude band monitoring allowed the follow up on the injury and the recovery. Results: We observed the appearance of two new emission bands more evident at the injury spectra when compared to the spectrums from normal non-exposed tissue. Following such spectral bands was possible to observe the establishment and recovery. Conclusions: The fluorescence spectroscopy is a promising technique in distinguishing between normal and UV induced skin change helping the evaluation of changes which are irreversible cancer tissue characteristics.

  17. Fluorescent Sensors for Biological Applications

    Directory of Open Access Journals (Sweden)

    Hui-wang Ai

    2014-09-01

    Full Text Available Fluorescence is one of the most important analytical methods used in biological studies. In the past decade or two, instrumentation in this field has greatly advanced, and now it is possible to detect single photons or fluorescent molecules [1,2], or break the Abbe diffraction limit to distinguish two points spaced less than 50 nm apart [3]. Concurrently, the development of improved fluorescent probes, which can be coupled with state-of-the-art instruments, has been equally important. This special issue on “fluorescent biosensors” in Sensors reports recent results from eight research groups in the field of sensor development. It includes three review articles, and six research articles reporting original results. [...

  18. Reversible logic gate using adiabatic superconducting devices

    Science.gov (United States)

    Takeuchi, N.; Yamanashi, Y.; Yoshikawa, N.

    2014-09-01

    Reversible computing has been studied since Rolf Landauer advanced the argument that has come to be known as Landauer's principle. This principle states that there is no minimum energy dissipation for logic operations in reversible computing, because it is not accompanied by reductions in information entropy. However, until now, no practical reversible logic gates have been demonstrated. One of the problems is that reversible logic gates must be built by using extremely energy-efficient logic devices. Another difficulty is that reversible logic gates must be both logically and physically reversible. Here we propose the first practical reversible logic gate using adiabatic superconducting devices and experimentally demonstrate the logical and physical reversibility of the gate. Additionally, we estimate the energy dissipation of the gate, and discuss the minimum energy dissipation required for reversible logic operations. It is expected that the results of this study will enable reversible computing to move from the theoretical stage into practical usage.

  19. X-ray fluorescence holography

    CERN Document Server

    Hayashi, K; Takahashi, Y

    2003-01-01

    X-ray fluorescence holography (XFH) is a new structural analysis method of determining a 3D atomic arrangement around fluorescing atoms. We developed an XFH apparatus using advanced X-ray techniques and succeeded in obtaining high-quality hologram data. Furthermore, we introduced applications to the structural analysis of a thin film and the environment around dopants and, discussed the quantitative analysis of local lattice distortion. (author)

  20. Vasectomy reversal : a clinical update

    NARCIS (Netherlands)

    A.P. Patel (Abhishek); R.P. Smith (Ryan)

    2016-01-01

    textabstractVasectomy is a safe and effective method of contraception used by 42-60 million men worldwide. Approximately 3%-6% of men opt for a vasectomy reversal due to the death of a child or divorce and remarriage, change in financial situation, desire for more children within the same marriage,

  1. CAPSULE REPORT: REVERSE OSMOSIS PROCESS

    Science.gov (United States)

    A failure analysis has been completed for the reverse osmosis (RO) process. The focus was on process failures that result in releases of liquids and vapors to the environment. The report includes the following: 1) A description of RO and coverage of the principles behind the proc...

  2. CAPSULE REPORT: REVERSE OSMOSIS PROCESS

    Science.gov (United States)

    A failure analysis has been completed for the reverse osmosis (RO) process. The focus was on process failures that result in releases of liquids and vapors to the environment. The report includes the following: 1) A description of RO and coverage of the principles behind the proc...

  3. A Framework for Reverse Logistics

    NARCIS (Netherlands)

    M.P. de Brito (Marisa); R. Dekker (Rommert)

    2003-01-01

    textabstractReverse Logistics has been stretching out worldwide, involving all the layers of supply chains in various industry sectors. While some actors in the chain have been forced to take products back, others have pro-actively done so, attracted by the value in used products One way or the othe

  4. Time-reversible Hamiltonian systems

    NARCIS (Netherlands)

    Schaft, Arjan van der

    1982-01-01

    It is shown that transfer matrices satisfying G(-s) = G(s) = G^T(-s) have a minimal Hamiltonian realization with an energy which is the sum of potential and kinetic energy, yielding the time reversibility of the equations. Furthermore connections are made with an associated gradient system. The

  5. Reversible colour change in Arthropoda.

    Science.gov (United States)

    Umbers, Kate D L; Fabricant, Scott A; Gawryszewski, Felipe M; Seago, Ainsley E; Herberstein, Marie E

    2014-11-01

    The mechanisms and functions of reversible colour change in arthropods are highly diverse despite, or perhaps due to, the presence of an exoskeleton. Physiological colour changes, which have been recorded in 90 arthropod species, are rapid and are the result of changes in the positioning of microstructures or pigments, or in the refractive index of layers in the integument. By contrast, morphological colour changes, documented in 31 species, involve the anabolism or catabolism of components (e.g. pigments) directly related to the observable colour. In this review we highlight the diversity of mechanisms by which reversible colour change occurs and the evolutionary context and diversity of arthropod taxa in which it has been observed. Further, we discuss the functions of reversible colour change so far proposed, review the limited behavioural and ecological data, and argue that the field requires phylogenetically controlled approaches to understanding the evolution of reversible colour change. Finally, we encourage biologists to explore new model systems for colour change and to engage scientists from other disciplines; continued cross-disciplinary collaboration is the most promising approach to this nexus of biology, physics, and chemistry.

  6. Reverse Knowledge Transfer in MNEs

    DEFF Research Database (Denmark)

    Mudambi, Ram; Piscitello, Lucia; Rabbiosi, Larissa

    2014-01-01

    , and that the curvilinearity is greater for greenfield entries relative to acquisition entries. The U-shaped relationship between subsidiary innovativeness and reverse knowledge transfers, as well as the sensitivity of this result to entry mode are important new findings in the literature on the role of subsidiaries...

  7. Fluorescence lifetimes: fundamentals and interpretations.

    Science.gov (United States)

    Noomnarm, Ulai; Clegg, Robert M

    2009-01-01

    Fluorescence measurements have been an established mainstay of photosynthesis experiments for many decades. Because in the photosynthesis literature the basics of excited states and their fates are not usually described, we have presented here an easily understandable text for biology students in the style of a chapter in a text book. In this review we give an educational overview of fundamental physical principles of fluorescence, with emphasis on the temporal response of emission. Escape from the excited state of a molecule is a dynamic event, and the fluorescence emission is in direct kinetic competition with several other pathways of de-excitation. It is essentially through a kinetic competition between all the pathways of de-excitation that we gain information about the fluorescent sample on the molecular scale. A simple probability allegory is presented that illustrates the basic ideas that are important for understanding and interpreting most fluorescence experiments. We also briefly point out challenges that confront the experimenter when interpreting time-resolved fluorescence responses.

  8. Fluorescence detection of esophageal neoplasia

    Science.gov (United States)

    Borisova, E.; Vladimirov, B.; Avramov, L.

    2008-06-01

    White-light endoscopy is well-established and wide used modality. However, despite the many technological advances that have been occurred, conventional endoscopy is suboptimal and usually detects advanced stage lesions. The limitations of standard endoscopy initiate development of spectroscopic techniques, additional to standard endoscopic equipment. One of the most sensitive approaches is fluorescence spectroscopy of gastrointestinal mucosa for neoplasia detection. In the recent study delta-aminolevulinic acid/Protoporphyrin IX (5-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus. The 5-ALA is administered per os six hours before measurements at dose 20 mg/kg weight. Excitation source has max of emission at 405 nm and light is delivered by the standard light guide of the endoscopic equipment. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to reabsorption of blood. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of 5-ALA/PpIX only in abnormal sites Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

  9. Characterization of excited-state reactions with instant spectra of fluorescence kinetics

    Energy Technology Data Exchange (ETDEWEB)

    Tomin, Vladimir I., E-mail: tomin@apsl.edu.pl; Ushakou, Dzmitryi V.

    2015-10-15

    Comprehensible knowledge of the excited-state proton transfer processes in organic compounds is overwhelmingly important not only for physics, but also chemistry and Life Sciences, since they play a key role in main processes of photosynthesis and functioning of biological organisms. Moreover compounds with Excited-State Intramolecular Proton Transfer (ESIPT) are in the focus of the interest of scientists throughout the world, because dual fluorescence spectra of such objects corresponding to two forms of molecular structure (normal and photoproduct) are very sensitive to characteristics of molecular microenvironment. This property allows to use such substances as fluorescent probes for diverse applications in chemistry and Life Sciences. But at the same time studying of proton transfer processes is not simple, because this process is characterized by extremely fast times (on picoseconds time scale and less order) and very often contribution of reverse reactions is essentially complicates an interpretation of observed properties of dual fluorescence. Hence, understanding of a role of reversible reactions is crucial for a comprehensive description of all processes accompanying excited state reactions. We discuss new approach for treatment ESIPT reaction on the basis of experimentally measured instant spectra of dual fluorescence and temporal behavior of ratiometric signal of normal to tautomer form intensities. Simple analytical expressions show in transparent way how to distinguish a degree of reverse reaction contribution to ratiometric signal. A validation of the approach under consideration is fulfilled with two different flavonols – 3-hydroxyflavone and 4′-(Dimethylamino)-3-hydroxyflavone – representing two extreme cases in affecting reversible reaction on dual emission. A comparing of new approach and traditional method when we analyze kinetics of separate the N* and T* fluorescence bands decays, has been carried out. - Highlights: • The excited

  10. Combining fluorescence and bioluminescence microscopy.

    Science.gov (United States)

    Goda, Kazuhito; Hatta-Ohashi, Yoko; Akiyoshi, Ryutaro; Sugiyama, Takashi; Sakai, Ikuko; Takahashi, Takeo; Suzuki, Hirobumi

    2015-08-01

    Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells.

  11. Tunable fluorescence enhancement based on bandgap-adjustable 3D Fe3O4 nanoparticles

    Science.gov (United States)

    Hu, Fei; Gao, Suning; Zhu, Lili; Liao, Fan; Yang, Lulu; Shao, Mingwang

    2016-06-01

    Great progress has been made in fluorescence-based detection utilizing solid state enhanced substrates in recent years. However, it is still difficult to achieve reliable substrates with tunable enhancement factors. The present work shows liquid fluorescence enhanced substrates consisting of suspensions of Fe3O4 nanoparticles (NPs), which can assemble 3D photonic crystal under the external magnetic field. The photonic bandgap induced by the equilibrium of attractive magnetic force and repulsive electrostatic force between adjacent Fe3O4 NPs is utilized to enhance fluorescence intensity of dye molecules (including R6G, RB, Cy5, DMTPS-DCV) in a reversible and controllable manner. The results show that a maximum of 12.3-fold fluorescence enhancement is realized in the 3D Fe3O4 NP substrates without the utilization of metal particles for PCs/DMTPS-DCV (1.0 × 10-7 M, water fraction (f w) = 90%).

  12. [Analyses of biogenic related compounds based on intramolecular excimer-forming fluorescence derivatization].

    Science.gov (United States)

    Yoshida, Hideyuki

    2003-08-01

    A highly selective and sensitive method based on a novel concept is introduced for the assay of biological substances. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, followed by reverse-phase HPLC. Polyamines, polyphenols, and dicarboxylic acids, which have two or more reactive functional groups in a molecule, were converted to the corresponding polypyrene-labeled derivatives by reaction with the appropriate pyrene reagent. The derivatives exhibited intramolecular excimer fluorescence (440-520 nm), which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted by pyrene reagents and monopyrene-labeled derivatives of monofunctional compounds. With excimer fluorescence detection, highly selective and sensitive determination of polyamines, polyphenols, and dicarboxylic acids can be achieved. Furthermore, the methods were successfully applied to the determination of various biological and environmental substances in real samples, which require only a small amount of sample and simple pretreatment.

  13. Interlocked DNA nanostructures controlled by a reversible logic circuit.

    Science.gov (United States)

    Li, Tao; Lohmann, Finn; Famulok, Michael

    2014-09-17

    DNA nanostructures constitute attractive devices for logic computing and nanomechanics. An emerging interest is to integrate these two fields and devise intelligent DNA nanorobots. Here we report a reversible logic circuit built on the programmable assembly of a double-stranded (ds) DNA [3]pseudocatenane that serves as a rigid scaffold to position two separate branched-out head-motifs, a bimolecular i-motif and a G-quadruplex. The G-quadruplex only forms when preceded by the assembly of the i-motif. The formation of the latter, in turn, requires acidic pH and unhindered mobility of the head-motif containing dsDNA nanorings with respect to the central ring to which they are interlocked, triggered by release oligodeoxynucleotides. We employ these features to convert the structural changes into Boolean operations with fluorescence labelling. The nanostructure behaves as a reversible logic circuit consisting of tandem YES and AND gates. Such reversible logic circuits integrated into functional nanodevices may guide future intelligent DNA nanorobots to manipulate cascade reactions in biological systems.

  14. Iodination increases the activity of verapamil derivatives in reversing PGP multidrug resistance.

    Science.gov (United States)

    Barattin, Regis; Gerby, Bastien; Bourges, Kevin; Hardy, Gaëlle; Olivares, Jose; Boutonnat, Jean; Arnoult, Christophe; D'Hardemare, Amaury D U Moulinet; Ronot, Xavier

    2010-07-01

    Iodinated derivatives of verapamil were synthesized and tested as P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) reversal agents. The ability of these compounds to revert MDR was evaluated on daunorubicin-resistant K562 cells, by measuring the intracellular accumulation of rhodamine 123, a fluorescent probe of Pgp transport activity. One of the investigated compounds (16c) was found to be a more potent MDR reversal agent than verapamil and cyclosporin A, used as reference molecules. Further in vitro studies showed that compound 16c restored daunorubicin activity and, when used alone, did not induce cell death, cell cycle perturbation and modification of calcium channel activity in comparison with verapamil.

  15. A reversible nanoconfined chemical reaction.

    Science.gov (United States)

    Nielsen, Thomas K; Bösenberg, Ulrike; Gosalawit, Rapee; Dornheim, Martin; Cerenius, Yngve; Besenbacher, Flemming; Jensen, Torben R

    2010-07-27

    Hydrogen is recognized as a potential, extremely interesting energy carrier system, which can facilitate efficient utilization of unevenly distributed renewable energy. A major challenge in a future "hydrogen economy" is the development of a safe, compact, robust, and efficient means of hydrogen storage, in particular, for mobile applications. Here we report on a new concept for hydrogen storage using nanoconfined reversible chemical reactions. LiBH4 and MgH2 nanoparticles are embedded in a nanoporous carbon aerogel scaffold with pore size Dmax approximately 21 nm and react during release of hydrogen and form MgB2. The hydrogen desorption kinetics is significantly improved compared to bulk conditions, and the nanoconfined system has a high degree of reversibility and stability and possibly also improved thermodynamic properties. This new scheme of nanoconfined chemistry may have a wide range of interesting applications in the future, for example, within the merging area of chemical storage of renewable energy.

  16. Reverse osmosis water purification system

    Science.gov (United States)

    Ahlstrom, H. G.; Hames, P. S.; Menninger, F. J.

    1986-01-01

    A reverse osmosis water purification system, which uses a programmable controller (PC) as the control system, was designed and built to maintain the cleanliness and level of water for various systems of a 64-m antenna. The installation operates with other equipment of the antenna at the Goldstone Deep Space Communication Complex. The reverse osmosis system was designed to be fully automatic; with the PC, many complex sequential and timed logic networks were easily implemented and are modified. The PC monitors water levels, pressures, flows, control panel requests, and set points on analog meters; with this information various processes are initiated, monitored, modified, halted, or eliminated as required by the equipment being supplied pure water.

  17. Reversible Watermarking Using Statistical Information

    Directory of Open Access Journals (Sweden)

    Kurugollu Fatih

    2010-01-01

    Full Text Available In most reversible watermarking methods, a compressed location map is exploited in order to ensure reversibility. Besides, in some methods, a header containing critical information is appended to the payload for the extraction and recovery process. Such schemes have a highly fragile nature; that is, changing a single bit in watermarked data may prohibit recovery of the original host as well as the embedded watermark. In this paper, we propose a new scheme in which utilizing a compressed location map is completely removed. In addition, the amount of auxiliary data is decreased by employing the adjacent pixels information. Therefore, in addition to quality improvement, independent authentication of different regions of a watermarked image is possible.

  18. Molecular Simulation of Reverse Micelles

    Science.gov (United States)

    Chowdhary, Janamejaya; Ladanyi, Branka

    2009-03-01

    Reverse micelles (RM) are surfactant assemblies containing a nanosized water pool dissolved in a hydrophobic solvent. Understanding their properties is crucial for insight into the effect of confinement on aqueous structure, dynamics as well as physical processes associated with solutes in confinement. We perform molecular dynamics simulations for the RM formed by the surfactant Aerosol-OT (AOT) in isooctane (2,2,4-trimethyl pentane) in order to study the effect of reverse micelle size on the aqueous phase. The structure of the RM is quantified in terms of the radial and pair density distributions. Dynamics are studied in terms of the mean squared displacements and various orientational time correlation functions in different parts of the RM so as to understand the effect of proximity to the interface on aqueous dynamics. Shape fluctuations of the RM are also analyzed.

  19. Chemoenzymatic reversible immobilization and labeling of proteins without prior purification.

    Science.gov (United States)

    Rashidian, Mohammad; Song, James M; Pricer, Rachel E; Distefano, Mark D

    2012-05-23

    Site-specific chemical modification of proteins is important for many applications in biology and biotechnology. Recently, our laboratory and others have exploited the high specificity of the enzyme protein farnesyltransferase (PFTase) to site-specifically modify proteins through the use of alternative substrates that incorporate bioorthogonal functionality including azides and alkynes. In this study, we evaluate two aldehyde-containing molecules as substrates for PFTase and as reactants in both oxime and hydrazone formation. Using green fluorescent protein (GFP) as a model system, we demonstrate that the purified protein can be enzymatically modified with either analogue to yield aldehyde-functionalized proteins. Oxime or hydrazone formation was then employed to immobilize, fluorescently label, or PEGylate the resulting aldehyde-containing proteins. Immobilization via hydrazone formation was also shown to be reversible via transoximization with a fluorescent alkoxyamine. After characterizing this labeling strategy using pure protein, the specificity of the enzymatic process was used to selectively label GFP present in crude E. coli extract followed by capture of the aldehyde-modified protein using hydrazide-agarose. Subsequent incubation of the immobilized protein using a fluorescently labeled or PEGylated alkoxyamine resulted in the release of pure GFP containing the desired site-specific covalent modifications. This procedure was also employed to produce PEGylated glucose-dependent insulinotropic polypeptide (GIP), a protein with potential therapeutic activity for diabetes. Given the specificity of the PFTase-catalyzed reaction coupled with the ability to introduce a CAAX-box recognition sequence onto almost any protein, this method shows great potential as a general approach for selective immobilization and labeling of recombinant proteins present in crude cellular extract without prior purification. Beyond generating site-specifically modified proteins, this

  20. Reversal agents in anaesthesia and critical care

    Directory of Open Access Journals (Sweden)

    Nibedita Pani

    2015-01-01

    Full Text Available Despite the advent of short and ultra-short acting drugs, an in-depth knowledge of the reversal agents used is a necessity for any anaesthesiologist. Reversal agents are defined as any drug used to reverse the effects of anaesthetics, narcotics or potentially toxic agents. The controversy on the routine reversal of neuromuscular blockade still exists. The advent of newer reversal agents like sugammadex have made the use of steroidal neuromuscular blockers like rocuronium feasible in rapid sequence induction situations. We made a review of the older reversal agents and those still under investigation for drugs that are regularly used in our anaesthesia practice.

  1. Gallstone identification by fluorescence spectroscopy

    Science.gov (United States)

    Pradhan, Asima; Laxmi, B. V.; Jena, Sidhartha S.; Khulbe, P. K.; Bist, Hari D.; Agarwal, Asha

    1998-04-01

    Gallstones have been classified as being cholesterol type and pigment type. The classification is important for diet control of the patient to avoid recurrence of the stone. Spectroscopy is a sensitive technique to determine the composition of the gallstone both in-vitro and in-vivo. this work deals with the fluorescence spectroscopy of gallstone. For fluorescence spectroscopic studies of gallstone, samples were excited with 5 mw of 488 nm line of argon-ion laser and spectra were recorded with a SPEX 1877E triplemate attached with a cooled PMT and DM3000R data acquisition system. Fluorescence spectra from pure cholesterol and bilirubin were also recorded for comparison. Different types of gallstones: mixed, cholesterol, pigment type were studied. All spectra exhibited a very broad band, 500 to 800 nm and sometimes two bands, depending on type of stone. Pure cholesterol shows three prominent fluorescence peaks at 513, 550, 583 nm along with two peaks at approximately 568 and 586 nm. Pure bilirubin shows prominent peak at 628 nm, without any Raman line. From fluorescence spectra different types of stones are identified. Different gallstones studied show a mixture of cholesterol and bilirubin types and the ratio of the two varies from one sample type to another.

  2. Plasmonics Enhanced Smartphone Fluorescence Microscopy

    KAUST Repository

    Wei, Qingshan

    2017-05-12

    Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

  3. Inhibition of telomerase with human telomerase reverse transcriptase antisense enhances tumor necrosis factor-a-induced apoptosis in bladder cancer cells

    Institute of Scientific and Technical Information of China (English)

    GAO Xiao-dong; CHEN Yi-rong

    2007-01-01

    Background Telomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells.Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-o (TNF-α)-induced apoptosis.Methods Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system.hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry.Results AS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-α while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group.Conclusions AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase

  4. CONCEPTUAL ISSUES REGARDING REVERSE LOGISTICS

    Directory of Open Access Journals (Sweden)

    Ioana Olariu

    2014-01-01

    Full Text Available As the power of consumers is growing, the product return for customer service and customer retention has become a common practice in the competitive market, which propels the recent practice of reverse logistics in companies. Many firms attracted by the value available in the flow, have proactively participated in handling returned products at the end of their usefulness or from other parts of the product life cycle. Reverse logistics is the flow and management of products, packaging, components and information from the point of consumption to the point of origin. It is a collection of practices similar to those of supply chain management, but in the opposite direction, from downstream to upstream. It involves activities such as reuse, repair, remanufacture, refurbish, reclaim and recycle. For the conventional forward logistics systems, the flow starts upstream as raw materials, later as manufactured parts and components to be assembled and continues downstream to reach customers as final products to be disposed once they reach their economic or useful lives. In reverse logistics, the disposed products are pushed upstream to be repaired, remanufactured, refurbished, and disassembled into components to be reused or as raw material to be recycled for later use.

  5. Reversible Simulations of Elastic Collisions

    CERN Document Server

    Perumalla, Kalyan S

    2013-01-01

    Consider a system of N identical hard spherical particles moving in a d-dimensional box and undergoing elastic, possibly multi-particle, collisions. We develop a new algorithm that recovers the pre-collision state from the post-collision state of the system, across a series of consecutive collisions, with essentially no memory overhead. The challenge in achieving reversibility for an n-particle collision (where, n << N) arises from the presence of nd-d-1 degrees of freedom during each collision, and from the complex geometrical constraints placed on the colliding particles. To reverse the collisions in a traditional simulation setting, all of the particular realizations of these degrees of freedom during the forward simulation must be saved. This limitation is addressed here by first performing a pseudo-randomization of angles, ensuring determinism in the reverse path for any values of n and d. To address the more difficult problem of geometrical and dynamic constraints, a new approach is developed whic...

  6. Reversible Oxidative Addition at Carbon.

    Science.gov (United States)

    Eichhorn, Antonius F; Fuchs, Sonja; Flock, Marco; Marder, Todd B; Radius, Udo

    2017-04-07

    The reactivity of N-heterocyclic carbenes (NHCs) and cyclic alkyl amino carbenes (cAACs) with arylboronate esters is reported. The reaction with NHCs leads to the reversible formation of thermally stable Lewis acid/base adducts Ar-B(OR)2 ⋅NHC (Add1-Add6). Addition of cAAC(Me) to the catecholboronate esters 4-R-C6 H4 -Bcat (R=Me, OMe) also afforded the adducts 4-R-C6 H4 Bcat⋅cAAC(Me) (Add7, R=Me and Add8, R=OMe), which react further at room temperature to give the cAAC(Me) ring-expanded products RER1 and RER2. The boronate esters Ar-B(OR)2 of pinacol, neopentylglycol, and ethyleneglycol react with cAAC at RT via reversible B-C oxidative addition to the carbene carbon atom to afford cAAC(Me) (B{OR}2 )(Ar) (BCA1-BCA6). NMR studies of cAAC(Me) (Bneop)(4-Me-C6 H4 ) (BCA4) demonstrate the reversible nature of this oxidative addition process.

  7. Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP.

    Science.gov (United States)

    Colletier, Jacques-Philippe; Sliwa, Michel; Gallat, François-Xavier; Sugahara, Michihiro; Guillon, Virginia; Schirò, Giorgio; Coquelle, Nicolas; Woodhouse, Joyce; Roux, Laure; Gotthard, Guillaume; Royant, Antoine; Uriarte, Lucas Martinez; Ruckebusch, Cyril; Joti, Yasumasa; Byrdin, Martin; Mizohata, Eiichi; Nango, Eriko; Tanaka, Tomoyuki; Tono, Kensuke; Yabashi, Makina; Adam, Virgile; Cammarata, Marco; Schlichting, Ilme; Bourgeois, Dominique; Weik, Martin

    2016-03-03

    Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins.

  8. Synthesis and Spectroscopic Investigation of Diketopyrrolopyrrole - Spiropyran Dyad for Fluorescent Switch Application.

    Science.gov (United States)

    Doddi, Siva; Narayanaswamy, K; Ramakrishna, Bheerappagari; Singh, Surya Prakash; Bangal, Prakriti Ranjan

    2016-11-01

    We report the synthesis and characterization of a new fluorescent dyad SP-DPP-SP(9) via efficient palladium-catalyzed Sonogashira coupling of prop-2-yn-1-yl 3-(3',3'dimethyl-6-nitrospiro[chromene-2,2'-indolin]-1'-yl)propanoatespiropyran, SP(8), a well known photochromic accepter, with 3,6-bis(5-bromothiophen-2-yl)-2,5-bis((R)-2-ethylhexyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione, DPP(4), a highly fluorescent donor. Under visible light exposure the SP unit is in a closed hydrophobic form, whereas under UV irradiation it converts to a polar, hydrophilic open form named Merocyanine (MC), which is responsible for functioning of photo-switch application. The photochemistry pertaining to fluorescence switch, 'on/off' behaviour, of model dyad SP-DPP-SP(9) is experimentally analyzed in solution as well as in solid state in polymer matrices by photoluminescence(PL) and absorption spectroscopy. After absorption of UV light the spiropyran unit of the dyad under goes the rupture of the spiro C-O bond leading to the formation of MC. The absorption band of MC fairly overlaps to the fluorescence of DPP unit resulting quenching of fluorescence via fluorescence resonance energy transfer from exited DPP unit to ground state MC. In contrary, the fluorescence of DPP is fully regained upon transformation of MC to SP by exposure to visible light or thermal stimuli. Hence, the fluorescence intensity of dyad 9 is regulated by reversible conversion among the two states of the photochromic spiropyran units and the fluorescence resonance energy transfer (FRET) between the MC form of SP and the DPP unit. Conversely, these scrutiny of the experiment express that the design of dyad 9 is viable as efficient fluorescent switch molecule in many probable commercial applications, such as, logic gates and photonic and optical communications.

  9. Fluorescence applications in molecular neurobiology.

    Science.gov (United States)

    Taraska, Justin W; Zagotta, William N

    2010-04-29

    Macromolecules drive the complex behavior of neurons. For example, channels and transporters control the movements of ions across membranes, SNAREs direct the fusion of vesicles at the synapse, and motors move cargo throughout the cell. Understanding the structure, assembly, and conformational movements of these and other neuronal proteins is essential to understanding the brain. Developments in fluorescence have allowed the architecture and dynamics of proteins to be studied in real time and in a cellular context with great accuracy. In this review, we cover classic and recent methods for studying protein structure, assembly, and dynamics with fluorescence. These methods include fluorescence and luminescence resonance energy transfer, single-molecule bleaching analysis, intensity measurements, colocalization microscopy, electron transfer, and bimolecular complementation analysis. We present the principles of these methods, highlight recent work that uses the methods, and discuss a framework for interpreting results as they apply to molecular neurobiology.

  10. Lasing from fluorescent protein crystals.

    Science.gov (United States)

    Oh, Heon Jeong; Gather, Malte C; Song, Ji-Joon; Yun, Seok Hyun

    2014-12-15

    We investigated fluorescent protein crystals for potential photonic applications, for the first time to our knowledge. Rod-shaped crystals of enhanced green fluorescent protein (EGFP) were synthesized, with diameters of 0.5-2 μm and lengths of 100-200 μm. The crystals exhibit minimal light scattering due to their ordered structure and generate substantially higher fluorescence intensity than EGFP or dye molecules in solutions. The magnitude of concentration quenching in EGFP crystals was measured to be about 7-10 dB. Upon optical pumping at 485 nm, individual EGFP crystals located between dichroic mirrors generated laser emission with a single-mode spectral line at 513 nm. Our results demonstrate the potential of protein crystals as novel optical elements for self-assembled, micro- or nano-lasers and amplifiers in aqueous environment.

  11. Evaluating Activated Carbon Adsorption of Dissolved Organic Matter and Micropollutants Using Fluorescence Spectroscopy.

    Science.gov (United States)

    Shimabuku, Kyle K; Kennedy, Anthony M; Mulhern, Riley E; Summers, R Scott

    2017-02-14

    Dissolved organic matter (DOM) negatively impacts granular activated carbon (GAC) adsorption of micropollutants and is a disinfection byproduct precursor. DOM from surface waters, wastewater effluent, and 1 kDa size fractions were adsorbed by GAC and characterized using fluorescence spectroscopy, UV-absorption, and size exclusion chromatography (SEC). Fluorescing DOM was preferentially adsorbed relative to UV-absorbing DOM. Humic-like fluorescence (peaks A and C) was selectively adsorbed relative to polyphenol-like fluorescence (peaks T and B) potentially due to size exclusion effects. In the surface waters and size fractions, peak C was preferentially removed relative to peak A, whereas the reverse was found in wastewater effluent, indicating that humic-like fluorescence is associated with different compounds depending on DOM source. Based on specific UV-absorption (SUVA), aromatic DOM was preferentially adsorbed. The fluorescence index (FI), if interpreted as an indicator of aromaticity, indicated the opposite but exhibited a strong relationship with average molecular weight, suggesting that FI might be a better indicator of DOM size than aromaticity. The influence of DOM intermolecular interactions on adsorption were minimal based on SEC analysis. Fluorescence parameters captured the impact of DOM size on the fouling of 2-methylisoborneol and warfarin adsorption and correlated with direct competition and pore blockage indicators.

  12. Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations.

    Science.gov (United States)

    Smith, Elizabeth M; Hennen, Jared; Chen, Yan; Mueller, Joachim D

    2015-07-01

    This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Furthermore, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C delta 1 labeled with green fluorescent protein on ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area-to-volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells.

    Science.gov (United States)

    Lubbeck, Jennifer L; Dean, Kevin M; Ma, Hairong; Palmer, Amy E; Jimenez, Ralph

    2012-05-01

    Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts; however, the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, nontemporally coincident excitation beams. Here, we characterize the design and operation of a cytometer with a three-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW to 179 kW/cm(2)). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: S(beam3)/S(beam1)) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multichannel fluorescence cytometry and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching.

  14. Going Viral with Fluorescent Proteins.

    Science.gov (United States)

    Costantini, Lindsey M; Snapp, Erik L

    2015-10-01

    Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

  15. Fluorescence for high school students

    CERN Document Server

    Schultheiss, Niek G

    2012-01-01

    In a not obligatory series of lessons for high school students in the Netherlands we discuss the fluorescence aspects of anthracene. These lessons were developed because HiSPARC (High school Project on Astrophysics Research with Cosmics) detection of cosmic rays are available for different secondary schools. With the help of special designed scintillator detection stations, containing anthracene, cosmic rays can be detected. Fluorescence of anthracene is one of the topics discussed in these series of extra curricular lessons aimed at excellent pupils working on cosmic radiation within the HiSPARC - project.

  16. Garbageless reversible implementation of integer linear transformations

    DEFF Research Database (Denmark)

    Burignat, Stéphane; Vermeirsch, Kenneth; De Vos, Alexis;

    2013-01-01

    Discrete linear transformations are important tools in information processing. Many such transforms are injective and therefore prime candidates for a physically reversible implementation into hardware. We present here reversible digital implementations of different integer transformations on fou...

  17. Ancient Magnetic Reversals: Clues to the Geodynamo.

    Science.gov (United States)

    Hoffman, Kenneth A.

    1988-01-01

    Discusses the question posed by some that the earth's magnetic field may reverse. States that rocks magnetized by ancient fields may offer clues to the underlying reversal mechanism in the earth's core. (TW)

  18. Reversible logic gate using adiabatic superconducting devices

    National Research Council Canada - National Science Library

    Takeuchi, N; Yamanashi, Y; Yoshikawa, N

    2014-01-01

    .... However, until now, no practical reversible logic gates have been demonstrated. One of the problems is that reversible logic gates must be built by using extremely energy-efficient logic devices...

  19. 14 CFR 33.97 - Thrust reversers.

    Science.gov (United States)

    2010-01-01

    ... Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Block Tests; Turbine Aircraft Engines § 33.97 Thrust reversers. (a) If the engine incorporates a reverser, the endurance calibration, operation, and vibration tests prescribed...

  20. SELECTED PROBLEMS OF REVERSE LOGISTICS IN POLAND

    OpenAIRE

    Agata Mesjasz-Lech

    2009-01-01

    This paper presents the essence of reverse logistics and directions of physical and information flows between logistic network partners. It also analyses effects of implementation of the principles of reverse logistics in Poland in the years 2004-2007

  1. Design of a High Performance Reversible Multiplier

    Directory of Open Access Journals (Sweden)

    Md.Belayet Ali

    2011-11-01

    Full Text Available Reversible logic circuits are increasingly used in power minimization having applications such as low power CMOS design, optical information processing, DNA computing, bioinformatics, quantum computing and nanotechnology. The problem of minimizing the number of garbage outputs is an important issue in reversible logic design. In this paper we propose a new 44 universal reversible logic gate. The proposed reversible gate can be used to synthesize any given Boolean functions. The proposed reversible gate also can be used as a full adder circuit. In this paper we have used Peres gate and the proposed Modified HNG (MHNG gate to construct the reversible fault tolerant multiplier circuit. We show that the proposed 44 reversible multiplier circuit has lower hardware complexity and it is much better and optimized in terms of number of reversible gates and number of garbage outputs with compared to the existing counterparts.

  2. Reversible Conformational Changes of PsbO Protein Detected by Terahertz Time-Domain Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    CHEN Hua; CHEN Gui-Ying; LI Shu-Qin; WANG Li

    2009-01-01

    We used a terahertz time-domain spectroscope (THz-TDS) to detect the reversible conformations2 changes of PsbO protein induced by N-bromosuccinimide and Guanidine Hydrochloride.The veracity and sensitivity are confirmed by the fluorescence emission spectra.The results demonstrate that THz-TDS has both advantages and disadvantages in monitoring the denaturation process of proteins,which is important in applying THz-TDS technique to studying biomolecules.

  3. Activity Regulation of Lignin Peroxidase from Phanerochaete chrysosporium in Nonionic Reversed Micellar Medium

    Institute of Scientific and Technical Information of China (English)

    Dan WANG; Xi Rong HUANG; Cai Xia LIU; Yue Zhong LI; Yin Bo QU; Pei Ji GAO

    2005-01-01

    The activity of lignin peroxidase (LiP) in reversed micelles of polyoxyethylene lauryl ether (Brij30) changed with the molar ratio of water to the surfactant and the denaturant concentration of guanidinium chloride. At low water contents the activity of LiP could be enhanced by the denaturant at moderate concentration. This phenomenon, together with the spectral characteristics of the intrinsic fluorescence of LiP, suggested that the conformation of the active center of LiP was flexible.

  4. Design of High speed Low Power Reversible Vedic multiplier and Reversible Divider

    Directory of Open Access Journals (Sweden)

    Srikanth G Department of Electronics & Communication Engineerig, Indur Institute of Engineering & Technology, Siddipet, Medak, JNTUH University, Telangana, India.

    2014-09-01

    Full Text Available This paper bring out a 32X32 bit reversible Vedic multiplier using "Urdhva Tiryakabhayam" sutra meaning vertical and crosswise, is designed using reversible logic gates, which is the first of its kind. Also in this paper we propose a new reversible unsigned division circuit. This circuit is designed using reversible components like reversible parallel adder, reversible left-shift register, reversible multiplexer, reversible n-bit register with parallel load line. The reversible vedic multiplier and reversible divider modules have been written in Verilog HDL and then synthesized and simulated using Xilinx ISE 9.2i. This reversible vedic multiplier results shows less delay and less power consumption by comparing with array multiplier.

  5. Statistical Learning, Letter Reversals, and Reading

    Science.gov (United States)

    Treiman, Rebecca; Gordon, Jessica; Boada, Richard; Peterson, Robin L.; Pennington, Bruce F.

    2014-01-01

    Reversal errors play a prominent role in theories of reading disability. We examined reversal errors in the writing of letters by 5- to 6-year-old children. Of the 130 children, 92 had a history of difficulty in producing speech sounds, a risk factor for reading problems. Children were more likely to reverse letter forms that face left, such as…

  6. Garbage collection for reversible functional languages

    DEFF Research Database (Denmark)

    Mogensen, Torben Ægidius

    2015-01-01

    Reversible languages are programming languages where all programs can run both forwards and backwards. Reversible functional languages have been proposed that use symmetric pattern matching and data construction. To be reversible, these languages require linearity: Every variable must be used exa...

  7. A functional language for describing reversible logic

    DEFF Research Database (Denmark)

    Thomsen, Michael Kirkedal

    2012-01-01

    . Reversibility of descriptions is guaranteed with a type system based on linear types. The language is applied to three examples of reversible computations (ALU, linear cosine transformation, and binary adder). The paper also outlines a design flow that ensures garbage- free translation to reversible logic...

  8. THEORETICAL FRAMES FOR DESIGNING REVERSE LOGISTICS PROCESSES

    OpenAIRE

    Grabara, Janusz K.; Sebastian Kot

    2009-01-01

    Logistics processes of return flow became more and more important in present business practice. Because of better customer satisfaction, environmental and financial aspects many enterprises deal with reverse logistics performance. The paper is a literature review focused on the design principles of reverse logistics processes Keywords: reverse logistics, designing.

  9. Dynamic Reverse Code Generation for Backward Execution

    DEFF Research Database (Denmark)

    Lee, Jooyong

    2007-01-01

    . In this paper, we present a method to generate reverse code, so that backtracking can be performed by executing reverse code. The novelty of our work is that we generate reverse code on-the-fly, while running a debugger, which makes it possible to apply the method even to debugging multi-threaded programs....

  10. Cleaning Our World through Reverse Graffiti

    Science.gov (United States)

    Randazzo, Gabe; LaJevic, Lisa

    2013-01-01

    Over the last decade artists have begun to experiment with "reverse pollution" techniques, such as reverse graffiti, which focuses on cleaning environmental surfaces. Having recently been introduced to the works of Moose, the artist known for inventing the reverse graffiti technique, the authors decided to design a curriculum to increase…

  11. REVERSE ENGINEERING AND ITS REALISTIC APPLICATIONS

    OpenAIRE

    Nikita Bakshi; Shruti Gujral

    2014-01-01

    Reverse-engineering is used for many purposes like as a learning tool, as a way to make compatible products that are cheaper than what is currently on the market. This paper discusses what is software, its type, reverse engineering, applications of reverse engineering and their tools.

  12. REVERSE ENGINEERING AND ITS REALISTIC APPLICATIONS

    Directory of Open Access Journals (Sweden)

    Nikita Bakshi

    2014-06-01

    Full Text Available Reverse-engineering is used for many purposes like as a learning tool, as a way to make compatible products that are cheaper than what is currently on the market. This paper discusses what is software, its type, reverse engineering, applications of reverse engineering and their tools.

  13. Highly Efficient Sky-Blue Fluorescent Organic Light Emitting Diode Based on Mixed Cohost System for Thermally Activated Delayed Fluorescence Emitter (2CzPN).

    Science.gov (United States)

    Sun, Jin Won; Kim, Kwon-Hyeon; Moon, Chang-Ki; Lee, Jeong-Hwan; Kim, Jang-Joo

    2016-04-20

    The mixed cohosts of 1,3-bis(N-carbazolyl)benzene and 2,8-bis(diphenylphosphoryl)dibenzothiophene have been developed for a highly efficient blue fluorescent oragnic light emitting diode (OLED) doped with a thermally activated delayed fluorescence (TADF) emitter [4,5-di (9H-carbazol-9-yl) phthalonitrile (2CzPN)]. We have demonstrated one of the highest external quantum efficiency of 21.8% in blue fluorescent OLEDs, which is identical to the theoretically achievable maximum electroluminescence efficiency using the emitter. Interestingly, the efficiency roll-off is large even under the excellent charge balance in the device and almost the same as the single host based devices, indicating that the efficiency roll-off in 2CzPN based TADF host is related to the material characteristics, such as low reverse intesystem crossing rate rather than charge imbalance.

  14. Remote Whispering Applying Time Reversal

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Brian Eric [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-07-16

    The purpose of this project was to explore the use of time reversal technologies as a means for communication to a targeted individual or location. The idea is to have the privacy of whispering in one’s ear, but to do this remotely from loudspeakers not located near the target. Applications of this work include communicating with hostages and survivors in rescue operations, communicating imaging and operational conditions in deep drilling operations, monitoring storage of spent nuclear fuel in storage casks without wires, or clandestine activities requiring signaling between specific points. This technology provides a solution in any application where wires and radio communications are not possible or not desired. It also may be configured to self calibrate on a regular basis to adjust for changing conditions. These communications allow two people to converse with one another in real time, converse in an inaudible frequency range or medium (i.e. using ultrasonic frequencies and/or sending vibrations through a structure), or send information for a system to interpret (even allowing remote control of a system using sound). The time reversal process allows one to focus energy to a specific location in space and to send a clean transmission of a selected signal only to that location. In order for the time reversal process to work, a calibration signal must be obtained. This signal may be obtained experimentally using an impulsive sound, a known chirp signal, or other known signals. It may also be determined from a numerical model of a known environment in which the focusing is desired or from passive listening over time to ambient noise.

  15. Biomolecule-to-fluorescent-color encoder: modulation of fluorescence emission via DNA structural changes.

    Science.gov (United States)

    Nishimura, Takahiro; Ogura, Yusuke; Yamada, Kenji; Ohno, Yuko; Tanida, Jun

    2014-07-01

    A biomolecule-to-fluorescent-color (B/F) encoder for optical readout of biomolecular information is proposed. In the B/F encoder, a set of fluorescence wavelengths and their intensity levels are used for coding of a biomolecular signal. A hybridization chain reaction of hairpin DNAs labeled with fluorescent reporters was performed to generate the fluorescence color codes. The fluorescence is modulated via fluorescence resonance energy transfer, which is controlled by DNA structural changes. The results demonstrate that fluorescent color codes can be configured based on two wavelengths and five intensities using the B/F encoder, and the assigned codes can be retrieved via fluorescence measurements.

  16. Fluorescence: a diagnostic tool for the detection of stress in plants

    Science.gov (United States)

    Chappelle, Emmett W.; Corp, Lawrence A.; McMurtrey, James E., III; Kim, Moon S.; Daughtry, Craig S. T.

    1997-01-01

    of excitation. Moreover, by comparing the ratio spectrum of a healthy versus nitrogen deficient leaf, one finds areas of crossover where trends can be completely reversed by changing excitation wavelength. As a result, the success of studies involving the measurement of chlorophyll a fluorescence depend greatly on the appropriate selection of excitation wavelength. Fluorescence sensing systems based on the above emission bands are being proposed or developed for ground based mobile vans, helicopters, and small aircraft. The goals of these efforts were to better define the origins of fluorescence and to improve our understanding of these light emissions in relationship to the physiological status of the plant.

  17. "Naked-eye" colorimetric and "turn-on" fluorometric chemosensors for reversible Hg2+ detection.

    Science.gov (United States)

    Wanichacheva, Nantanit; Praikaew, Panida; Suwanich, Thanapat; Sukrat, Kanjarat

    2014-01-24

    Two new Hg(2+)-colorimetric and fluorescent sensors based on 2-[3-(2-aminoethylsulfanyl) propylsulfanyl]ethanamine covalently bound to one and two units of rhodamine-6G moieties, 1 and 2, were synthesised, and their sensing behaviors toward metal ions were investigated by UV/Vis and fluorescence spectroscopy. Upon the addition of Hg(2+), the sensors exhibited highly sensitive "turn-on" fluorescence enhancement as well as a color change from colorless to pink, which was readily noticeable for naked eye detection. Especially, 1 exhibited the reversible behavior and revealed a very high selectivity in the presence of competitive ions, particularly Cu(2+), Ag(+), Pb(2+), Ca(2+), Cd(2+), Co(2+), Fe(2+), Mn(2+), Na(+), Ni(2+), K(+), Ba(2+), Li(+) and Zn(2+), with a low detection limit of 1.7 ppb toward Hg(2+).

  18. Reverse Triangle Inequalities for Potentials

    OpenAIRE

    Pritsker, I. E.; Saff, E. B.

    2013-01-01

    We study the reverse triangle inequalities for suprema of logarithmic potentials on compact sets of the plane. This research is motivated by the inequalities for products of supremum norms of polynomials. We find sharp additive constants in the inequalities for potentials, and give applications of our results to the generalized polynomials. We also obtain sharp inequalities for products of norms of the weighted polynomials $w^nP_n, deg(P_n)\\le n,$ and for sums of suprema of potentials with ex...

  19. Presbycusis: reversible with anesthesia drugs?

    Science.gov (United States)

    Kocher, Carl A

    2009-02-01

    Age-related hearing impairment, or presbycusis, is a degenerative condition not currently treatable by medication. It is therefore significant that the author, as a patient, experienced a reversal of high-frequency hearing loss during a 2-day period following abdominal surgery with general anesthesia. This report documents the surgery and the subsequent restoration of hearing, which was bilateral and is estimated to have exceeded 50dB at 4kHz. A possible role is noted for anesthetic agents such as lidocaine, propofol, or fentanyl. This experience may hold a clue for research toward the development of medical treatments for presbycusis.

  20. How to play Reverse Hex

    DEFF Research Database (Denmark)

    Toft, Bjarne; Hayward, Ryan B.; Henderson, Philip

    2012-01-01

    We present new results on how to play Reverse Hex, also known as Rex, or Misère Hex, on n × n boards. We give new proofs – and strengthened versions – of Lagarias and Sleator’s theorem (for n × n boards, each player can prolong the game until the board is full, so the first/second player can always......, we find second-player winning replies. Finally, in response to comments by Martin Gardner, for each n ≤ 5, we give a simple winning strategy for the n × n board....

  1. Fluorescence for high school students

    NARCIS (Netherlands)

    Schultheiss, N.G.; Kool, T.W.

    2012-01-01

    In a not obligatory series of lessons for high school students in the Netherlands we discuss the fluorescence aspects of anthracene. These lessons were developed because HiSPARC (High school Project on Astrophysics Research with Cosmics) detection of cosmic rays are available for different secondary

  2. Development of Sealed Fluorescence Spectrometer

    Institute of Scientific and Technical Information of China (English)

    QIAN; Hong-juan; ZHANG; Li-hua; LIU; Huan-liang; FAN; De-jun

    2012-01-01

    <正>In nuclear fuel reprocessing, the fluorescent analytical instrument can be used to analyze various trace elements, such as boron and thorium in uranium product. Due to the high radioactivity, strong acidity, fatal toxic and complex components of spent nuclear fuel reprocessing sample, analytical works become more difficult and instruments used can be damaged easier.

  3. A fluorescent probe for ecstasy.

    Science.gov (United States)

    Masseroni, D; Biavardi, E; Genovese, D; Rampazzo, E; Prodi, L; Dalcanale, E

    2015-08-18

    A nanostructure formed by the insertion in silica nanoparticles of a pyrene-derivatized cavitand, which is able to specifically recognize ecstasy in water, is presented. The absence of effects from interferents and an efficient electron transfer process occurring after complexation of ecstasy, makes this system an efficient fluorescent probe for this popular drug.

  4. Studying Photosynthesis by Measuring Fluorescence

    Science.gov (United States)

    Sanchez, Jose Francisco; Quiles, Maria Jose

    2006-01-01

    This paper describes an easy experiment to study the absorption and action spectrum of photosynthesis, as well as the inhibition by heat, high light intensity and the presence of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the photosynthetic process. The method involves measuring the chlorophyll fluorescence emitted by intact…

  5. In vivo fluorescence lifetime tomography

    Science.gov (United States)

    Nothdurft, Ralph E.; Patwardhan, Sachin V.; Akers, Walter; Ye, Yunpeng; Achilefu, Samuel; Culver, Joseph P.

    2009-03-01

    Local molecular and physiological processes can be imaged in vivo through perturbations in the fluorescence lifetime (FLT) of optical imaging agents. In addition to providing functional information, FLT methods can quantify specific molecular events and multiplex diagnostic and prognostic information. We have developed a fluorescence lifetime diffuse optical tomography (DOT) system for in vivo preclinical imaging. Data is captured using a time-resolved intensified charge coupled device (ICCD) system to measure fluorescence excitation and emission in the time domain. Data is then converted to the frequency domain, and we simultaneously reconstruct images of yield and lifetime using an extension to the normalized Born approach. By using differential phase measurements, we demonstrate DOT imaging of short lifetimes (from 350 ps) with high precision (+/-5 ps). Furthermore, this system retains the efficiency, speed, and flexibility of transmission geometry DOT. We demonstrate feasibility of FLT-DOT through a progressive series of experiments. Lifetime range and repeatability are first measured in phantoms. Imaging of subcutaneous implants then verifies the FLT-DOT approach in vivo in the presence of inhomogeneous optical properties. Use in a common research scenario is ultimately demonstrated by imaging accumulation of a targeted near-infrared (NIR) fluorescent-labeled peptide probe (cypate-RGD) in a mouse with a subcutaneous tumor.

  6. Fluorescence diagnostics in oncological gynecology

    Science.gov (United States)

    Belyaeva, Ludmila A.; Adamyan, Leila V.; Kozachenko, Vladimir P.; Stratonnikov, Alexander A.; Stranadko, Eugene F.; Loschenov, Victor B.

    2003-10-01

    The method of fluorescent diagnostics (FD) of tumors is a promising tool that may allow to increase sensitivity of tumor detection especially at initial stages. One of the most promising photosensitizers today is 5-aminolevulinic acid (5-ALA) that, actually, is not photosensitizer itself but precursor of protoporphyrin IX (PpIX). This paper deals with cancer diagnostics in gynecology by means of ALA-induced Pp IX laser-fluorescence spectroscopy. The tissue fluorescence spectra in vivo were studied in patients with various pathologies of ovaries, uterine and vulva after 5-aminolevulinic acid administration. It was shown that different pathologies varies in accumulation of Pp IX. Coefficient of fluorescence kf for normal tissue is not high, but exceptions are endometrium and mucous membrane of uterine tubes. Benign tumors of uterus and ovary have low values of kf, but polyps of endometrium exhibit high kf. Optical express-biopsy is important for diagnosis of ovarian cancer and micrometastatic spread. Coefficients of diagnostic contrast were determined for cancer of endometrium, cervical cancer, vulvar cancer.

  7. Fluorescent Labeling of Nanometer Hydroxyapatite

    Institute of Scientific and Technical Information of China (English)

    Yuan ZHANG; Yuan YUAN; Changsheng LIU

    2008-01-01

    A novel surface treatment method using 3-aminopropyltriethoxysilane (AMPTES), was developed to immobilize the fluorescein molecule on nano-HAP (nanometer hydroxyapatite) powders. By pretreating the nano-HAP powders surface with AMPTES, fluorescein, chosen on the basis of the chemical structure of the nano- HAP powders, could be bound to the nano-HAP powders surface. The chemical compositions of nano-HAP before and after being labeled were characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The morphology, phase composition, and the fluorescence characteristics of the nano-HAP powders with and without staining were also investigated. The FTIR and XPS results revealed that fiuorescein had been successfully immobilized on the surface of AMPTES-bound nano-HAP powders via the acylamide bond formation between the -COOH of fluorescein and the -NH2 of AMPTES. The labeled nano-HAP powders possessed strong fluorescent intensity with a little deviation from the maximum emission wavelength of fluorescein. But the morphology and phase composition had no obvious alteration. Under fluorescence microscopy, the labeled nano-HAP powders., even after 24 h cell incubation, exhibited strong fluorescence.

  8. High-pressure SANS and fluorescence unfolding study of calmodulin.

    Science.gov (United States)

    Gibrat, Gabriel; Hoa, Gaston Hui Bon; Craescu, Constantin T; Assairi, Liliane; Blouquit, Yves; Annighöfer, Burkhard; May, Roland P; Bellissent-Funel, Marie-Claire

    2014-09-01

    Apo-calmodulin, a small soluble mainly α protein, is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80°C, whereas the calcium-saturated form is stable up to temperatures as high as 100°C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al., BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000bar (300MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form have been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease have been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.

  9. In Vitro Evaluation of Fluorescence Glucose Biosensor Response

    Directory of Open Access Journals (Sweden)

    Mamdouh Aloraefy

    2014-07-01

    Full Text Available Rapid, accurate, and minimally-invasive glucose biosensors based on Förster Resonance Energy Transfer (FRET for glucose measurement have the potential to enhance diabetes control. However, a standard set of in vitro approaches for evaluating optical glucose biosensor response under controlled conditions would facilitate technological innovation and clinical translation. Towards this end, we have identified key characteristics and response test methods, fabricated FRET-based glucose biosensors, and characterized biosensor performance using these test methods. The biosensors were based on competitive binding between dextran and glucose to concanavalin A and incorporated long-wavelength fluorescence dye pairs. Testing characteristics included spectral response, linearity, sensitivity, limit of detection, kinetic response, reversibility, stability, precision, and accuracy. The biosensor demonstrated a fluorescence change of 45% in the presence of 400 mg/dL glucose, a mean absolute relative difference of less than 11%, a limit of detection of 25 mg/dL, a response time of 15 min, and a decay in fluorescence intensity of 72% over 30 days. The battery of tests presented here for objective, quantitative in vitro evaluation of FRET glucose biosensors performance have the potential to form the basis of future consensus standards. By implementing these test methods for a long-visible-wavelength biosensor, we were able to demonstrate strengths and weaknesses with a new level of thoroughness and rigor.

  10. Fluorescence imaging-based high-throughput screening of fast- and slow-cycling LOV proteins.

    Directory of Open Access Journals (Sweden)

    Fuun Kawano

    Full Text Available Light-oxygen-voltage (LOV domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from Avena sativa phototropin 1 (AsLOV2. The gene library was introduced into Escherichia coli cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3 × 10(3 s (78-fold slower than wild-type AsLOV2. The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.

  11. Fluorescent sensors based on bacterial fusion proteins

    Science.gov (United States)

    Prats Mateu, Batirtze; Kainz, Birgit; Pum, Dietmar; Sleytr, Uwe B.; Toca-Herrera, José L.

    2014-06-01

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

  12. Optimized reversible binary-coded decimal adders

    DEFF Research Database (Denmark)

    Thomsen, Michael Kirkedal; Glück, Robert

    2008-01-01

    their design. The optimized 1-decimal BCD full-adder, a 13 × 13 reversible logic circuit, is faster, and has lower circuit cost and less garbage bits. It can be used to build a fast reversible m-decimal BCD full-adder that has a delay of only m + 17 low-power reversible CMOS gates. For a 32-decimal (128-bit...... in reversible logic design by drastically reducing the number of garbage bits. Specialized designs benefit from support by reversible logic synthesis. All circuit components required for optimizing the original design could also be synthesized successfully by an implementation of an existing synthesis algorithm...

  13. Reversing invasion in bistable systems.

    Science.gov (United States)

    Alzahrani, Ebraheem O; Davidson, Fordyce A; Dodds, Niall

    2012-12-01

    In this paper, we discuss a class of bistable reaction-diffusion systems used to model the competitive interaction of two species. The interactions are assumed to be of classic "Lotka-Volterra" type and we will consider a particular problem with relevance to applications in population dynamics: essentially, we study under what conditions the interplay of relative motility (diffusion) and competitive strength can cause waves of invasion to be halted and reversed. By establishing rigorous results concerning related degenerate and near-degenerate systems, we build a picture of the dependence of the wave speed on system parameters. Our results lead us to conjecture that this class of competition model has three "zones of response". In the central zone, varying the motility can slow, halt and reverse invasion. However, in the two outer zones, the direction of invasion is independent of the relative motility and is entirely determined by the relative competitive strengths. Furthermore, we conjecture that for a large class of competition models of the type studied here, the wave speed is an increasing function of the relative motility.

  14. Reverse Genetic Approaches in Zebrafish

    Institute of Scientific and Technical Information of China (English)

    Peng Huang; Zuoyan Zhu; Shuo Lin; Bo Zhang

    2012-01-01

    Zebrafish (Danio rerio) is a well-established vertebrate animal model.A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism.Morpholino is the most widely used reagent to knock down target gene expression post-transcriptionally.For a long time,targeted genome modification has been heavily relied on large-scale traditional forward genetic screens,such as ENU (N-ethyl-N-nitrosourea) mutagenesis derived TILLING (Targeting Induced Local Lesions IN Genomes)strategy and pseudo-typed retrovirus mediated insertional mutagenesis.Recently,engineered endonucleases,including ZFNs (zinc finger nucleases) and TALENs (transcription activator-like effector nucleases),provide new and efficient strategies to directly generate sitespecific indel mutations by inducing double strand breaks in target genes.Here we summarize the major reverse genetic approaches for loss-of-function studies used and emerging in zebrafish,including strategies based on genome-wide mutagenesis and methods for sitespecific gene targeting.Future directions and expectations will also be discussed.

  15. Catalytic performance and thermostability of chloroperoxidase in reverse micelle: achievement of a catalytically favorable enzyme conformation.

    Science.gov (United States)

    Wang, Yali; Wu, Jinyue; Ru, Xuejiao; Jiang, Yucheng; Hu, Mancheng; Li, Shuni; Zhai, Quanguo

    2011-06-01

    The catalytic performance of chloroperoxidase (CPO) in peroxidation of 2, 2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) and oxidation of indole in a reverse micelle composed of surfactant-water-isooctane-pentanol was investigated and optimized in this work. Some positive results were obtained as follows: the peroxidation activity of CPO was enhanced 248% and 263%, while oxidation activity was enhanced 215% and 222% in cetyltrimethylammonium bromide (CTABr) reverse micelle medium and dodecyltrimethylammonium bromide (DTABr) medium, respectively. Thermostability was also greatly improved in reverse micelle: at 40 °C, CPO essentially lost all its activity after 5 h incubation, while 58-76% catalytic activity was retained for both reactions in the two reverse micelle media. At 50 °C, about 44-75% catalytic activity remained for both reactions in reverse micelle after 2 h compared with no observed activity in pure buffer under the same conditions. The enhancement of CPO activity was dependent mainly on the surfactant concentration and structure, organic solvent ratio (V(pentanol)/V(isooctane)), and water content in the reverse micelle. The obtained kinetic parameters showed that the catalytic turnover frequency (k(cat)) was increased in reverse micelle. Moreover, the lower K(m) and higher k(cat)/K(m) demonstrated that both the affinity and specificity of CPO to substrates were improved in reverse micelle media. Fluorescence, circular dichroism (CD) and UV-vis spectra assays indicated that a catalytically favorable conformation of enzyme was achieved in reverse micelle, including the strengthening of the protein α-helix structure, and greater exposure of the heme prosthetic group for easy access of the substrate in bulk solution. These results are promising in view of the industrial applications of this versatile biological catalyst.

  16. Online Testable Decoder using Reversible Logic

    Directory of Open Access Journals (Sweden)

    Hemalatha. K. N. Manjula B. B. Girija. S

    2012-02-01

    Full Text Available The project proposes to design and test 2 to 4 reversible Decoder circuit with arbitrary number of gates to an online testable reversible one and is independent of the type of reversible gate used. The constructed circuit can detect any single bit errors and to convert a decoder circuit that is designed by reversible gates to an online testable reversible decoder circuit. Conventional digital circuits dissipate a significant amount of energy because bits of information are erased during the logic operations. Thus if logic gates are designed such that the information bits are not destroyed, the power consumption can be reduced. The information bits are not lost in case of a reversible computation. Reversible logic can be used to implement any Boolean logic function.

  17. [Feature abstraction and spectral reconstruction of three-dimensional fluorescence spectra of oil in water].

    Science.gov (United States)

    Tian, Guang-Jun

    2008-04-01

    In the present paper, spectral reconstruction of three-dimensional fluorescence spectra of oil was studied based on singular value division (SVD) of fluorescence excitation-emission matrix (EEM). Depending on oil components, three-dimensional (3-D) fluorescence spectra of oils can be seen as their "fingerprints". Feature abstraction and selection of 3-D fluorescence spectra is important to oil identification. Statistic parameters such as the average, standard error, centroid, kurtosis, geometrical distribution as well as main-shaft slope selected, forming "apparent statistic feature" vector of 3-D fluorescence spectra of oils, are limited due to their roughness. And the apparent statistic feature vector can not support spectral reconstruction reversely. In this paper, with singular-value features abstracted from EEMs composing the feature chain, reversal spectral reconstruction can be realized, and its information loss can be estimated. In other words, a pan-gene series of 3-D fluorescence spectra consists of singular values and their corresponding accompanying vectors. By SVD operation on EEMs of dozens of oil samples in water, their singular values accompanied with corresponding vectors were calculated showing obviously gathering energy distribution. With singular values being properly cut, principal feature parameters were selected and combined with their accompanying vectors composing the pan-gene series (or singular-value pan-gene chain) and spectral reconstruction was reversely completed. A couple of spectra (original and reconstructed) of diesel as the typical oil in water were presented for comparison. It can be seen that there is no obvious difference between the reconstructed and original spectra. Certainly, information loss exists but in an scheduled extent. It is shown that the singular-value features of fluorescence excitation-emission matrix (EEM) represent spectral gathering energy, and the length-limited pan-gene chain has the ability to reconstruct

  18. NOVEL FLUORESCENT PROBES FOR THE DOPAMINE TRANSPORTER

    DEFF Research Database (Denmark)

    Cha, J; Vægter, Christian Bjerggaard; Adkins, Erica

    To enable visualization of the dopamine transporter (DAT) through fluorescence technologies we have synthesized a novel series of fluorescently tagged analogs of cocaine. Previous structure-activity relationship (SAR) studies have demonstrated that the dopamine transporter (DAT) can tolerate...

  19. NOVEL FLUORESCENT PROBES FOR THE DOPAMINE TRANSPORTER

    DEFF Research Database (Denmark)

    Cha, J; Vægter, Christian Bjerggaard; Adkins, Erica

    To enable visualization of the dopamine transporter (DAT) through fluorescence technologies we have synthesized a novel series of fluorescently tagged analogs of cocaine. Previous structure-activity relationship (SAR) studies have demonstrated that the dopamine transporter (DAT) can tolerate...

  20. Fluorescent Protein Approaches in Alpha Herpesvirus Research

    Directory of Open Access Journals (Sweden)

    Ian B. Hogue

    2015-11-01

    Full Text Available In the nearly two decades since the popularization of green fluorescent protein (GFP, fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1 and pseudorabies virus (PRV structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

  1. Preparation and Application of Fluorescent Carbon Dots

    Directory of Open Access Journals (Sweden)

    Jun Zuo

    2015-01-01

    Full Text Available Fluorescent carbon dots (CDs are a novel type of fluorescent nanomaterials, which not only possess the specific quantum confinement effects of nanomaterials due to the small size of nanomaterials, but also have good biocompatibility and high fluorescence. Meanwhile, fluorescence CDs overcome the shortcomings of high toxicity of traditional nanomaterials. Moreover, the preparation procedure of fluorescent CDs is simple and easy. Therefore, fluorescent CDs have great potential applied in photocatalysis, biochemical sensing, bioimaging, drug delivery, and other related areas. In this paper, recent hot researches on fluorescent CDs are reviewed and some problems in the progress of fluorescent CDs are also summarized. At last, a future outlook in this direction is presented.

  2. Photochromicity and fluorescence lifetimes of green fluorescent protein

    OpenAIRE

    1999-01-01

    The green fluorescent protein (GFP) of the bioluminescent jellyfish Aequorea and its mutants have gained widespread usage as an indicator of structure and function within cells. Proton transfer has been implicated in the complex photophysics of the wild-type molecule, exhibiting a protonated A species excited at 400 nm, and two deprotonated excited-state species I* and B* with red-shifted excitation similar to 475 nm. Photochromicity between the protonated and deprotonated species has been re...

  3. EML4-ALK Fusion Lung Cancer: A Rare Acquired Event

    Directory of Open Access Journals (Sweden)

    Sven Perner

    2008-03-01

    Full Text Available A recurrent gene fusion between EML4 and ALK in 6.7% of non-small cell lung cancers (NSCLCs and NKX2-1 (TTF1, TITF1 high-level amplifications in 12% of adenocarcinomas of the lung were independently reported recently. Because the EML4-ALK fusion was only shown by a reverse transcription-polymerase chain reaction approach, we developed fluorescent in situ hybridization assays to interrogate more than 600 NSCLCs using break-apart probes for EML4 and ALK. We found that EML4-ALK fusions occur in less than 3% of NSCLC samples and that EML4 and/or ALK amplifications also occur. We also observed that, in most cases in which an EML4/ALK alteration is detected, not all of the tumor cells harbor the lesion. By using a detailed multi-fluorescent in situ hybridization probe assay and reverse transcription-polymerase chain reaction, we have evidence that other, more common mechanisms besides gene inversion exist including the possibility of other fusion partners for ALK and EML4. Furthermore, we confirmed the NKX2-1 high-level amplification in a significant subset of NSCLC and found this amplification to be mutually exclusive to ALK and EML4 rearrangements.

  4. Clinical significance of CK19 mRNA detection in peripheral blood of cervical cancer patients by fluorescent quantitative RT-PCR%荧光定量逆转录聚合酶链式反应检测宫颈癌患者外周血CK19mRNA表达的临床意义

    Institute of Scientific and Technical Information of China (English)

    寿华锋; 倪镌; 陈雅卿; 孙海燕

    2011-01-01

    目的 探讨宫颈癌患者外周血中CK19mRNA的表达及其临床意义.方法 应用荧光定量逆转录聚合酶链式反应(FQ-RT-PCR)技术检测138例经病理证实的宫颈癌及36例妇科良性肿瘤(对照组)外周血液中CK19mRNA的表达,并分析其与临床病理因素的关系.结果 138例宫颈癌组外周血CK19阳性率(69.6%)高于对照组(13.9%,x2=36.34,P=0.000).宫颈癌组中早期宫颈癌(Ⅰ-ⅡA期)外周血CK19 mRNA阳性率(57.9%)低于中晚期宫颈癌(Ⅱb-Ⅳ期,80.6%,x2=8.14,P=0.004).早期宫颈癌患者CK19 mRNA的表达与年龄(<40岁、≥40岁)、肿瘤大小(<4cm、≥≥4 cm)、病理类型(鳞癌、非鳞癌)、浸润深度、淋巴转移(+/-)均无关(P>0.05);与肿瘤分期(ⅠA、ⅠB、ⅡA)、组织分化(G1 -2、G3)、脉管瘤栓(+/-)有关(x2=9.59,7.27,9.94,P<0.01).早期宫颈癌患者Logistica多因素分析示CK19 mRNA的表达与脉管瘤栓相关(x2 =5.29,P=0.021).结论 荧光定量RT-PCR技术可检测出各期宫颈癌患者外周血中CK19的表达,其敏感性和特异性高,可作为检测宫颈癌微转移的生物学指标,对判断宫颈癌的预后具有一定指导意义.%Objective To detect the expression of Cytokeratin 19 (CK19) mRNA in the peripheral blood of cervical carcinoma patients and evaluate its clinical significance.Methods The expression of CK19 mRNA was evaluated by fluorescent quantitative reverse transcription polymerase chain reaction ( FQRT-PCR ) in the peripheral blood of 138 patients with cervical carcinoma and 36 patients with benign gynecological tumors.In 138 patients,possible correlations between clinical pathological factors were analyzed.Results The positive expression rates of CK19 mRNA was 69.6% in 138 cervical carcinomas in comparison with 13.9% in benign gynecological tumors,and 57.9% in patients with FIGO Stage Ⅰ A to Ⅱ A cervical carcinoma in comparison with 80.6% in patients with FIGO Stage Ⅱb to Ⅳ cervical carcinoma

  5. Synthesis and characterization of new fluorescent nanoparticles

    Institute of Scientific and Technical Information of China (English)

    Liang Tao; Xu Hun; Zhu Jun Zhang

    2008-01-01

    A novel kind of fluorescent nanoparticles (FNPs) has been prepared using a precipitation polymerization method.Methacrylic acid,trimethylolpropane trimethacrylate and azobisisobutyronitrile were used as functional-monomer,cross-linker and initiator,respectively.Compared with other fluorescent nanoparticles,the FNPs have the characteristics including low dye leakage and good photostability.The fluorescence microscopy imaging indicates that the FNPs can be used as fluorescent labels in bioanalysis.

  6. Laser-Stimulated Fluorescence in Paleontology

    OpenAIRE

    Kaye, Thomas G.; Falk, Amanda R.; Michael Pittman; Sereno, Paul C.; Martin, Larry D.; Burnham, David A.; Enpu Gong; Xing Xu; Yinan Wang

    2015-01-01

    Fluorescence using ultraviolet (UV) light has seen increased use as a tool in paleontology over the last decade. Laser-stimulated fluorescence (LSF) is a next generation technique that is emerging as a way to fluoresce paleontological specimens that remain dark under typical UV. A laser's ability to concentrate very high flux rates both at the macroscopic and microscopic levels results in specimens fluorescing in ways a standard UV bulb cannot induce. Presented here are five paleontological c...

  7. Highlights of the optical highlighter fluorescent proteins.

    Science.gov (United States)

    Patterson, G H

    2011-07-01

    The development of super-resolution microscopy techniques using molecular localization, such as photoactivated localization microscopy, fluorescence photoactivated localization microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy with independent running acquisition and many others, has heightened interest in molecules that will be grouped here into a category referred to as 'optical highlighter' fluorescent proteins. This review will survey many of the advances in development of fluorescent proteins for optically highlighting sub-populations of fluorescently labelled molecules.

  8. A dual-stimuli-responsive fluorescent switch ultrathin film

    Science.gov (United States)

    Li, Zhixiong; Liang, Ruizheng; Liu, Wendi; Yan, Dongpeng; Wei, Min

    2015-10-01

    Stimuli-responsive fluorescent switches have shown broad applications in optical devices, biological materials and intelligent responses. Herein, we describe the design and fabrication of a dual-stimuli-responsive fluorescent switch ultrathin film (UTF) via a three-step layer-by-layer (LBL) technique: (i) encapsulation of spiropyran (SP) within an amphiphilic block copolymer (PTBEM) to give the (SP@PTBEM) micelle; (ii) the mixture of riboflavin (Rf) and poly(styrene 4-sulfonate) (PSS) to enhance the adhesion ability of small molecules; (iii) assembly of negatively charged SP@PTBEM and Rf-PSS with cationic layered double hydroxide (LDH) nanoplatelets to obtain the (Rf-PSS/LDH/SP@PTBEM)n UTFs (n: bilayer number). The assembly process of the UTFs and their luminescence properties, as monitored by fluorescence spectroscopy and scanning electron microscopy (SEM), present a uniform and ordered layered structure with stepwise growth. The resulting Rf-PSS/LDH/SP@PTBEM UTF serves as a three-state switchable multicolor (green, yellow, and red) luminescent system based on stimulation from UV/Vis light and pH, with an acceptable reversibility. Therefore, this work provides a facile way to fabricate stimuli-responsive solid-state film switches with tunable-color luminescence, which have potential applications in the areas of displays, sensors, and rewritable optical memory and fluorescent logic devices.Stimuli-responsive fluorescent switches have shown broad applications in optical devices, biological materials and intelligent responses. Herein, we describe the design and fabrication of a dual-stimuli-responsive fluorescent switch ultrathin film (UTF) via a three-step layer-by-layer (LBL) technique: (i) encapsulation of spiropyran (SP) within an amphiphilic block copolymer (PTBEM) to give the (SP@PTBEM) micelle; (ii) the mixture of riboflavin (Rf) and poly(styrene 4-sulfonate) (PSS) to enhance the adhesion ability of small molecules; (iii) assembly of negatively charged SP

  9. Quantification of fluorescent reporters in plant cells.

    Science.gov (United States)

    Pound, Michael; French, Andrew P; Wells, Darren M

    2015-01-01

    Fluorescent reporters are powerful tools for plant research. Many studies require accurate determination of fluorescence intensity and localization. Here, we describe protocols for the quantification of fluorescence intensity in plant cells from confocal laser scanning microscope images using semiautomated software and image analysis techniques.

  10. Demonstrating Fluorescence with Neon Paper and Plastic

    Science.gov (United States)

    Birriel, Jennifer J.; Roe, Clarissa

    2015-01-01

    Several papers in this journal have dealt with the fluorescence in orange neon plastic, olive oil, and soda. In each case, the fluorescent emission was excited by either green or violet-blue laser light. In this paper, we examine the fluorescent emission spectra of so-called neon colored papers and plastic clipboards available in department and…

  11. Characterization of natural fluorescence in mice

    Science.gov (United States)

    Djeziri, Salim; Ma, Guobin; Mincu, Niculae; Benyamin Seeyar, Anader; Khayat, Mario

    2008-02-01

    One important challenge for in-vivo imaging fluorescence in cancer research and related pharmaceutical studies is to discriminate the exogenous fluorescence signal of the specific tagged agents from the natural fluorescence. For mice, natural fluorescence is composed of endogenous fluorescence from organs like the skin, the bladder, etc. and from ingested food. The discrimination between the two kinds of fluorescence makes easy monitoring the targeted tissues. Generally, the amplitude of the fluorescence signal depends on the location and on the amount of injected fluorophore, which is limited in in-vivo experiments. This paper exposes some results of natural fluorescence analysis from in-vivo mice experiments using a time domain small animal fluorescence imaging system: eXplore Optix TM. Fluorescence signals are expressed by a Time Point Spread Function (TPSF) at each scan point. The study uses measures of similarity applied purposely to the TPSF to evaluate the discrepancy and/or the homogeneity of scanned regions of a mouse. These measures allow a classification scheme to be performed on the TPSF's based on their temporal shapes. The work ends by showing how the exogenous fluorescence can be distinguished from natural fluorescence by using the TPSF temporal shape.

  12. Demonstrating Fluorescence with Neon Paper and Plastic

    Science.gov (United States)

    Birriel, Jennifer J.; Roe, Clarissa

    2015-01-01

    Several papers in this journal have dealt with the fluorescence in orange neon plastic, olive oil, and soda. In each case, the fluorescent emission was excited by either green or violet-blue laser light. In this paper, we examine the fluorescent emission spectra of so-called neon colored papers and plastic clipboards available in department and…

  13. Molecular-sized fluorescent nanodiamonds

    Science.gov (United States)

    Vlasov, Igor I.; Shiryaev, Andrey A.; Rendler, Torsten; Steinert, Steffen; Lee, Sang-Yun; Antonov, Denis; Vörös, Márton; Jelezko, Fedor; Fisenko, Anatolii V.; Semjonova, Lubov F.; Biskupek, Johannes; Kaiser, Ute; Lebedev, Oleg I.; Sildos, Ilmo; Hemmer, Philip. R.; Konov, Vitaly I.; Gali, Adam; Wrachtrup, Jörg

    2014-01-01

    Doping of carbon nanoparticles with impurity atoms is central to their application. However, doping has proven elusive for very small carbon nanoparticles because of their limited availability and a lack of fundamental understanding of impurity stability in such nanostructures. Here, we show that isolated diamond nanoparticles as small as 1.6 nm, comprising only ~400 carbon atoms, are capable of housing stable photoluminescent colour centres, namely the silicon vacancy (SiV). Surprisingly, fluorescence from SiVs is stable over time, and few or only single colour centres are found per nanocrystal. We also observe size-dependent SiV emission supported by quantum-chemical simulation of SiV energy levels in small nanodiamonds. Our work opens the way to investigating the physics and chemistry of molecular-sized cubic carbon clusters and promises the application of ultrasmall non-perturbative fluorescent nanoparticles as markers in microscopy and sensing.

  14. Ruby fluorescence pressure scale: Revisited

    Science.gov (United States)

    Liu, Lei; Bi, Yan; Xu, Ji-An

    2013-05-01

    Effect of non-hydrostatic stress on X-ray diffraction in a diamond anvil cell (DAC) is studied. The pressure gradient in the sample chamber leads to the broadening of the diffraction peaks, which increase with the hkl index of the crystal. It is found that the difference between the determined d-spacing compressive ratio d/d0 and the real d-spacing compressive ratio dr/d0 is determined by the yield stress of the pressure transmitting media (if used) and the shear modulus of the sample. On the basis of the corrected experiment data of Mao et al. (MXB86), which was used to calibrate the most widely used ruby fluorescence scale, a new relationship of ruby fluorescence pressure scale is corrected, i.e., P = (1904/9.827)[(1 + Δλ/λ0)9.827-1].

  15. New Fluorescence Probes for Biomolecules

    Directory of Open Access Journals (Sweden)

    Katarzyna Jurek

    2015-07-01

    Full Text Available Steady state fluorescence measurements have been used for the investigation of interaction between the bovine serum albumin (BSA and fluorescence probes: 3-hydroxy-2,4- bis[(3-methyl-1,3-benzoxazol-2(3H-ylidenemethyl]cyclobut-2-en-1-one (SQ6, 3-hydroxy- 2,4-bis[(3-methyl-1,3-benzothiazol-2(3H-ylidenemethyl]cyclobut-2-en-1-one (SQ7 and 3-hydroxy-2,4-bis[(1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidenemethyl]cyclobut-2-en-1-one (SQ8. The binding constant between bovine serum albumin and squarine dyes has been determined by using both the Benesi-Hildebrand and Stern-Volmer equations. The negative value of free energy change indicates the existence of a spontaneous complexation process of BSA with squarine dyes.

  16. Fluorescence spectroscopy for neoplasms control

    Science.gov (United States)

    Bratchenko, I. A.; Kristoforova, Yu. A.; Myakinin, O. O.; Artemyev, D. N.; Kozlov, S. V.; Moryatov, A. A.; Zakharov, V. P.

    2016-04-01

    Investigation of malignant skin tumors diagnosis was performed involving two setups for native tissues fluorescence control in visible and near infrared regions. Combined fluorescence analysis for skin malignant melanomas and basal cell carcinomas was performed. Autofluorescence spectra of normal skin and oncological pathologies stimulated by 457 nm and 785 nm lasers were registered for 74 skin tissue samples. Spectra of 10 melanomas and 27 basal cell carcinomas were registered ex vivo. Skin tumors analysis was made on the basis of autofluorescence spectra intensity and curvature for analysis of porphyrins, lipo-pigments, flavins and melanin. Separation of melanomas and basal cell carcinomas was performed on the basis of discriminant analysis. Overall accuracy of basal cell carcinomas and malignant melanomas separation in current study reached 86.5% with 70% sensitivity and 92.6% specificity.

  17. Sorting fluorescent nanocrystals with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Gerion, Daniele; Parak, Wolfgang J.; Williams, Shara C.; Zanchet, Daniela; Micheel, Christine M.; Alivisatos, A. Paul

    2001-12-10

    Semiconductor nanocrystals with narrow and tunable fluorescence are covalently linked to oligonucleotides. These biocompounds retain the properties of both nanocrystals and DNA. Therefore, different sequences of DNA can be coded with nanocrystals and still preserve their ability to hybridize to their complements. We report the case where four different sequences of DNA are linked to four nanocrystal samples having different colors of emission in the range of 530-640 nm. When the DNA-nanocrystal conjugates are mixed together, it is possible to sort each type of nanoparticle using hybridization on a defined micrometer -size surface containing the complementary oligonucleotide. Detection of sorting requires only a single excitation source and an epifluorescence microscope. The possibility of directing fluorescent nanocrystals towards specific biological targets and detecting them, combined with their superior photo-stability compared to organic dyes, opens the way to improved biolabeling experiments, such as gene mapping on a nanometer scale or multicolor microarray analysis.

  18. Multi Spectral Fluorescence Imager (MSFI)

    Science.gov (United States)

    Caron, Allison

    2016-01-01

    Genetic transformation with in vivo reporter genes for fluorescent proteins can be performed on a variety of organisms to address fundamental biological questions. Model organisms that may utilize an ISS imager include unicellular organisms (Saccharomyces cerevisiae), plants (Arabidopsis thaliana), and invertebrates (Caenorhabditis elegans). The multispectral fluorescence imager (MSFI) will have the capability to accommodate 10 cm x 10 cm Petri plates, various sized multi-well culture plates, and other custom culture containers. Features will include programmable temperature and light cycles, ethylene scrubbing (less than 25 ppb), CO2 control (between 400 ppm and ISS-ambient levels in units of 100 ppm) and sufficient airflow to prevent condensation that would interfere with imaging.

  19. FAMOUS. The fluorescence telescope prototype

    Energy Technology Data Exchange (ETDEWEB)

    Schumacher, Johannes; Bretz, Thomas; Hebbeker, Thomas; Lauscher, Markus; Middendorf, Lukas; Niggemann, Tim; Peters, Christine; Sommer, Dominik; Stephan, Maurice [III. Physikalisches Institut A, RWTH Aachen University (Germany); Auffenberg, Jan; Schaufel, Merlin [III. Physikalisches Institut B, RWTH Aachen University (Germany)

    2015-07-01

    One of the most successful techniques for the detection of air showers produced by ultra-high-energy cosmic rays are fluorescence telescopes. The light produced by de-exciting nitrogen in the atmosphere is typically detected by photomultiplier tubes (PMTs). This technique has been successfully used by the Pierre Auger Observatory in Argentina for many years. Silicon photomultipliers (SiPMs) promise higher photon detection efficiencies than PMTs. This and other advantages motivate the construction of the fluorescence telescope prototype FAMOUS (First Auger Multi-pixel photon counter camera for the Observation of Ultra-high-energy air Showers) which makes use of SiPMs. In this talk we discuss the FAMOUS telescope with a new 64-pixel camera including power supply and DAQ.

  20. Correlative fluorescence and electron microscopy.

    Science.gov (United States)

    Schirra, Randall T; Zhang, Peijun

    2014-10-01

    Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associated with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology has led to rapid improvement in the protocols and has ushered in a new generation of instruments to reach the next level of correlation--integration.